Inhibition of bacterial biofilms with aryl carbamates

ABSTRACT

Disclosure is provided for carbamate compounds that prevent, remove and/or inhibit the formation of biofilms, compositions including these compounds, devices including these compounds, and methods of using the same.

RELATED APPLICATIONS

This application is a continuation of PCT Patent Application No.PCT/US2011/042941, filed Jul. 5, 2011, which claims the benefit under 35U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/361,654,filed Jul. 6, 2010, the contents of each of which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to compounds, compositions and methods useful forcontrolling biofilms.

BACKGROUND OF THE INVENTION

Biofilms are complex communities of microorganisms that are commonlyfound on a variety of substrates or surfaces that are moist or submerged(Musk et al., Curr. Med. Chem., 2006, 13, 2163; Donlan et al., Clin.Microbiol. Rev., 2002, 15, 167). Though primarily populated by bacteria,biofilms can also contain many different individual types ofmicroorganisms, e.g., bacteria, archaea, protozoa and algae. Theformation of biofilms can be thought of as a developmental process inwhich a few free-swimming (planktonic) bacteria adhere to a solidsurface and, in response to appropriate signals, initiate the formationof a complex sessile microcolony existing as a community of bacteria andother organisms. Bacteria within biofilms are usually embedded within amatrix, which can consist of protein, polysaccharide, nucleic acids, orcombinations of these macromolecules. The matrix is a critical featureof the biofilm that protects the inhabiting organisms from antiseptics,microbicides, and host cells. It has been estimated that bacteria withinbiofilms are upwards of 1.000-fold more resistant to conventionalantibiotics (Rasmussen et al., Int. J. Med. Microbiol., 2006, 296, 149).

Biofilms play a significant role in infectious disease. It is estimatedthat biofilms account for between 50-80% of microbial infections in thebody, and that the cost of these infections exceeds $1 billion annually.For example, persistent infections of indwelling medical devices remaina serious problem for patients, because eradication of these infectionsis virtually impossible. A few diseases in which biofilms have beenimplicated include endocarditis, otitis media, chronic prostatitis,periodontal disease, chronic urinary tract infections, and cysticfibrosis. The persistence of biofilm populations is linked to theirinherent insensitivity to antiseptics, antibiotics, and otherantimicrobial compounds or host cells.

Deleterious effects of biofilms are also found in non-medical settings.For example, biofilms are a major problem in the shipping industry.Biofilms form on and promote the corrosion of ship hulls and alsoincrease the roughness of the hulls, increasing the drag on the shipsand thereby increasing fuel costs. The biofilms can also promote theattachment of larger living structures, such as barnacles, to the hull.Fuel can account for half of the cost of marine shipping, and the lossin fuel efficiency due to biofilm formation is substantial. One methodof controlling biofilms is to simply scrape the films off of the hulls.However, this method is costly and time-consuming, and can promote thespread of troublesome non-native species in shipping waters. Anothermethod involves the use of antifouling coatings containing tin. However,tin-based coatings are now disfavored due to toxicity concerns.

Given the breadth of detrimental effects caused by bacterial biofilms,there has been an effort to develop small molecules that will inhibittheir formation (Musk et al., Curr. Med. Chem., 2006, 13, 2163). Theunderlying principle is that if bacteria can be maintained in theplanktonic state, they will either not attach to a target surface and/orthey can be killed by a lower dose of microbicide.

Despite the extent of biofilm driven problems, examples of structuralscaffolds that inhibit biofilm formation are rare (Musk et al., Curr.Med. Chem., 2006, 13, 2163). The few known examples include thehomoserine lactones (Geske et al., J. Am. Chem. Soc., 2005, 127, 12762),which are naturally-occurring bacterial signaling molecules thatbacteria use in quorum sensing (Dong et al., J. Microbiol., 2005, 43,101; Nealson et al., J. Bacteriol., 1970, 104, 313), brominatedfuranones isolated from the macroalga Delisea pulchra (Hentzer et al.,Microbiology-Sgm, 2002, 148, 87), and ursene triterpenes from the plantDiospyros dendo (Hu et al., J. Nat. Prod., 2006, 69, 118).

In addition, bacteria have an unparalleled ability to overcome foreignchemical insult. For example, resistance to vancomycin, “the antibioticof last resort,” has become more prevalent, and strains ofvancomycin-resistant Staphylococcus aureus have become a serious healthrisk. It has been predicted that it is simply a matter of time beforedifferent bacterial strains develop vancomycin resistance, and thesafety net that vancomycin has provided for decades in antibiotictherapy will no longer be available. Therefore, the identification ofchemical architectures useful to inhibit biofilm development is needed.

Because of their natural resistance to antibiotics, phagocytic cells,and other biocides, biofilms are difficult, if not impossible, toeradicate. Therefore, the identification of compounds that controlbiofilms is of critical need.

SUMMARY OF THE INVENTION

Provided herein are compounds of Formula (I):

wherein:

R¹ is an aryl, an amine-substituted aryl, or R¹ is a heteroaryl havingat least one nitrogen atom;

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl;

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl; and

R³ is alkyl, substituted cycloalkyl or unsubstituted cycloalkyl,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments of Formula (I), R¹ is selected from the groupconsisting of:

In some embodiments of Formula (I), the compound is a compound ofFormula (I)(a):

wherein:

R¹ is an amine-substituted aryl, or R¹ is a heteroaryl having at leastone nitrogen atom;

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl; and

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments of Formula (I)(a), R¹ is selected from the groupconsisting of:

In some embodiments of Formula (I), the compound is a compound ofFormula (I)(a)(i):

wherein:

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl; and

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments of Formula (I), the compound is a compound ofFormula (I)(a)(ii):

wherein:

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl; and

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments of Formula (I), the compound is a compound ofFormula (I)(a)(iii):

wherein:

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl; and

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments of Formula (I), the compound is selected from thegroup consisting of:

or a pharmaceutically acceptable salt or prodrug thereof.

Biofilm inhibiting compositions are provided, which include a carrierand an effective amount of a compound disclosed herein. Compositions arealso provided that include a compound disclosed herein in apharmaceutically acceptable carrier.

Compositions are further provided that include a compound disclosedherein covalently coupled to a substrate. In some embodiments, thesubstrate includes a polymeric material. In some embodiments, thesubstrate includes a solid support. In some embodiments, the substrateincludes a drainpipe, glaze ceramic, porcelain, glass, metal, wood,chrome, plastic, vinyl, and Formica® brand laminate (The DillerCorporation, Cincinnati, Ohio). In some embodiments, the substrateincludes shower curtains or liners, upholstery, laundry, and carpeting.

Biofilm inhibiting coating compositions are provided, including: (a) afilm-forming resin; (b) a solvent that disperses said resin; (c) aneffective amount of the compounds or compositions disclosed herein,wherein said effective amount inhibits the growth of a biofilm thereon;and (d) optionally, at least one pigment. In some embodiments, thecompound is covalently coupled to the resin. In some embodiments, theresin includes a polymeric material.

Substrates coated with coating composition disclosed herein are alsoprovided. In some embodiments, the substrate includes a polymericmaterial. In some embodiments, the substrate includes a solid support.In some embodiments, the substrate includes a drainpipe, glaze ceramic,porcelain, glass, metal, wood, chrome, plastic or polymeric material,vinyl, and laminates thereof such as Formica® brand laminate. In someembodiments, the substrate includes shower curtains or liners,upholstery, laundry, and carpeting.

Methods of controlling biofilm formation on a substrate are provided,including the step of contacting the substrate with a compound and/orcomposition disclosed herein in an amount effective to inhibit biofilmformation. In some embodiments, the substrate may include a drainpipe,glaze ceramic, porcelain, glass, metal, wood, chrome, plastic orpolymeric material, vinyl, and laminates thereof such as Formica® brandlaminate. In some embodiments, the biofilm includes Gram-positivebacteria.

Methods for treating and/or preventing a bacterial infection in asubject in need thereof are provided, including administering to saidsubject a compound and/or composition disclosed herein in an amounteffective to inhibit a biofilm component of said bacterial infection.

Also provided are medical devices, including (a) a medical devicesubstrate; and (b) an effective amount of a compound disclosed herein,either coating the substrate, or incorporated into the substrate,wherein said effective amount inhibits the growth of a biofilm thereon.In some embodiments, the medical device substrate may include stents,fasteners, ports, catheters, scaffolds and grafts. In some embodiments,the compound is covalently coupled to said substrate.

Compounds and/or compositions for use in a method to control a biofilmare further provided. Also provided is the use of compounds and/orcompositions disclosed herein for the preparation of a medicament forthe treatment and/or prevention of a bacterial infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1P illustrate the inhibitory effect of test compounds on S.aureus, MRSA, and E. coli biofilm formation.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention is further described below. All patent referencesreferred to in this patent application are hereby incorporated byreference in their entirety as if set forth fully herein.

A. DEFINITIONS

“Carbamate” refers to the commonly known moiety:

“H” refers to a hydrogen atom. “C” refers to a carbon atom. “N” refersto a nitrogen atom. “O” refers to an oxygen atom. “Halo” refers to F,Cl, Br or I. The term “hydroxy,” as used herein, refers to an —OHmoiety. “Br” refers to a bromine atom. “Cl” refers to a chlorine atom.“I” refers to an iodine atom. “F” refers to a fluorine atom.

An “acyl group” is intended to mean a group —C(O)—R, where R is asuitable substituent (for example, an acetyl group, a propionyl group, abutyroyl group, a benzoyl group, or an alkylbenzoyl group).

“Alkyl,” as used herein, refers to a straight or branched chainhydrocarbon containing from 1 or 2 to 10 or 20 or more carbon atoms(e.g., C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15,etc.). In some embodiments the alkyl can be a lower alkyl. “Lower alkyl”refers to straight or branched chain alkyl having from 1 to 3, or from 1to 5, or from 1 to 8 carbon atoms. Representative examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

As generally understood by those of ordinary skill in the art,“saturation” refers to the state in which all available valence bonds ofan atom (e.g., carbon) are attached to other atoms. Similarly,“unsaturation” refers to the state in which not all the availablevalence bonds are attached to other atoms; in such compounds the extrabonds usually take the form of double or triple bonds (usually withcarbon). For example, a carbon chain is “saturated” when there are nodouble or triple bonds present along the chain or directly connected tothe chain (e.g., a carbonyl), and is “unsaturated” when at least onedouble or triple bond is present along the chain or directly connectedto the chain (e.g., a carbonyl). Further, the presence or absence of asubstituent depending upon chain saturation will be understood by thoseof ordinary skill in the art to depend upon the valence requirement ofthe atom or atoms to which the substituent binds (e.g., carbon).

“Alkenyl,” as used herein, refers to a straight or branched chainhydrocarbon containing from 1 or 2 to 10 or 20 or more carbons, andcontaining at least one carbon-carbon double bond, formed structurally,for example, by the replacement of two hydrogens. Representativeexamples of “alkenyl” include, but are not limited to, ethenyl,2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl,2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl and the like.

“Alkynyl,” as used herein, refers to a straight or branched chainhydrocarbon group containing from 1 or 2 to 10 or 20 or more carbonatoms, and containing at least one carbon-carbon triple bond.Representative examples of alkynyl include, but are not limited, toacetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 1-butynyl andthe like.

The term “cycloalkyl,” as used herein, refers to a saturated cyclichydrocarbon group containing from 3 to 8 carbons or more. Representativeexamples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, cycloalkylgroups as described herein are optionally substituted (e.g., from 1 to 3or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, etc.

As understood in the art, the term “optionally substituted” indicatesthat the specified group is either unsubstituted, or substituted by oneor more suitable substituents. A “substituent” that is “substituted” isan atom or group which takes the place of a hydrogen atom on the parentchain or cycle of an organic molecule.

“Heterocyclo,” as used herein, refers to a monocyclic, bicyclic ortricyclic ring system. Monocyclic heterocycle ring systems areexemplified by any 5 or 6 member ring containing 1, 2, 3, or 4heteroatoms independently selected from the group consisting of: O, N,and S. The 5 member ring has from 0 to 2 double bonds, and the 6 memberring has from 0-3 double bonds. Representative examples of monocyclicring systems include, but are not limited to, azetidine, azepine,aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan,imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline,isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine,oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline,oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole,pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole,pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine,tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole,thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholinesulfone, sulfoxide, thiopyran, triazine, triazole, trithiane, and thelike. Bicyclic ring systems are exemplified by any of the abovemonocyclic ring systems fused to an aryl group as defined herein, acycloalkyl group as defined herein, or another monocyclic ring system asdefined herein. Representative examples of bicyclic ring systems includebut are not limited to, for example, benzimidazole, benzothiazole,benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole,benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3-benzodioxole,cinnoline, indazole, indole, indoline, indolizine, naphthyridine,isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline,phthalazine, pyranopyridine, quinoline, quinolizine, quinoxaline,quinazoline, tetrahydroisoquinoline, tetrahydroquinoline,thiopyranopyridine, and the like. In some embodiments, heterocyclogroups as described herein are optionally substituted (e.g., from 1 to 3or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, etc.

“Aryl” as used herein refers to a ring system having one or morearomatic rings. Representative examples of aryl include azulenyl,indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.The aryl groups of this invention can be substituted with 1, 2, 3, 4, or5 substituents independently selected from alkenyl, alkenyloxy, alkoxy,alkoxyalkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy,alkylsulfinyl, alkylsulfonyl, alkylthio, alkynyl, aryl, aryloxy, azido,arylalkoxy, arylalkyl, aryloxy, carboxy, cyano, formyl, halogen,haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, mercapto, nitro, sulfamyl,sulfo, sulfonate, —NR′R″ (wherein, R′ and R″ are independently selectedfrom hydrogen, alkyl, alkylcarbonyl, aryl, arylalkyl and formyl), and—C(O)NR′R″ (wherein R′ and R″ are independently selected from hydrogen,alkyl, alkylcarbonyl, aryl, arylalkyl, and formyl).

“Heteroaryl” means a cyclic, aromatic hydrocarbon in which one or morecarbon atoms have been replaced with heteroatoms (e.g., N, O or S). Ifthe heteroaryl group contains more than one heteroatom, the heteroatomsmay be the same or different. Examples of heteroaryl groups includepyridyl, pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, indolyl, isoindolyl,indolizinyl, triazolyl, pyridazinyl, indazolyl, purinyl, quinolizinyl,isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl,isothiazolyl, and benzo[b]thienyl. Preferred heteroaryl groups are fiveand six membered rings and contain from one to three heteroatomsindependently selected from the group consisting of: O, N, and S. Theheteroaryl group, including each heteroatom, can be unsubstituted orsubstituted with from 1 to 4 suitable substituents, as chemicallyfeasible. For example, the heteroatom S may be substituted with one ortwo oxo groups, which may be shown as ═O.

“Alkoxy,” as used herein, refers to an alkyl group, as defined herein,appended to the parent molecular moiety through an oxy group, as definedherein. Representative examples of alkoxy include, but are not limitedto, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy,hexyloxy and the like.

An “amine” or “amino” is intended to mean the group —NH₂.

An “amide” as used herein refers to an organic functional group having acarbonyl group (C═O) linked to a nitrogen atom (N), or a compound thatcontains this group, generally depicted as:

wherein, R and R′ can independently be any covalently-linked atom oratoms.

A “thiol” or “mercapto” refers to an —SH group or to its tautomer ═S.

A “sulfone” as used herein refers to a sulfonyl functional group,generally depicted as:

wherein, R can be any covalently-linked atom or atoms.

A “sulfoxide” as used herein refers to a sulfinyl functional group,generally depicted as:

wherein, R can be any covalently-linked atom or atoms.

The term “oxo,” as used herein, refers to a ═O moiety. The term “oxy,”as used herein, refers to a —O— moiety.

“Nitro” refers to the organic compound functional group —NO₂.

“Carbonyl” is a functional group having a carbon atom double-bonded toan oxygen atom (—C═O). “Carboxy” as used herein refers to a —COOHfunctional group, also written as —CO₂H or —(C═O)—OH.

A “pharmaceutically acceptable salt” is intended to mean a salt thatretains the biological effectiveness of the free acids and bases of aspecified compound and that is not biologically or otherwiseundesirable. Examples of pharmaceutically acceptable salts includesulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates,citrates, lactates, γ-hydroxybutyrates, glycollates, tartrates,methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, and mandelates.

A “prodrug” is intended to mean a compound that is converted underphysiological conditions or by solvolysis or metabolically to aspecified compound that is pharmaceutically active. A thoroughdiscussion is provided in T. Higuchi and V. Stella, Prodrugs as Noveldelivery Systems, Vol. 14 of the A.C.S. Symposium Series and in EdwardB. Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated by reference herein in their entirety.

B. ACTIVE COMPOUNDS

Active compounds are provided below. In some of the embodiments providedin the present invention, active compounds are carbamates. Activecompounds as described herein can be prepared as detailed below or inaccordance with known procedures or variations thereof that will beapparent to those skilled in the art.

As will be appreciated by those of skill in the art, the activecompounds of the various formulas disclosed herein may contain chiralcenters, e.g. asymmetric carbon atoms. Thus, the present invention isconcerned with the synthesis of both: (i) racemic mixtures of the activecompounds, and (ii) enantiomeric forms of the active compounds. Theresolution of racemates into enantiomeric forms can be done inaccordance with known procedures in the art. For example, the racematemay be converted with an optically active reagent into a diastereomericpair, and the diastereomeric pair subsequently separated into theenantiomeric forms.

Geometric isomers of double bonds and the like may also be present inthe compounds disclosed herein, and all such stable isomers are includedwithin the present invention unless otherwise specified. Also includedin active compounds of the invention are tautomers (e.g., tautomers oftriazole and/or imidazole) and rotamers. All chains defined by theformulas herein which include three or more carbons may be saturated orunsaturated unless otherwise indicated.

Provided herein are active compounds of Formula (I):

wherein:

R¹ is an aryl, an amine-substituted aryl, or R¹ is a heteroaryl havingat least one nitrogen atom;

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl;

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl; and

R³ is alkyl, substituted cycloalkyl or unsubstituted cycloalkyl,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments, R¹ is an amine-substituted aryl, or R¹ is aheteroaryl having at least one nitrogen atom.

In some embodiments, R³ is substituted or unsubstituted cycloalkyl.

In some embodiments of Formula (I), R³ is a substituted cycloalkylrepresented by Formula (I)(a):

wherein:

R¹ is an amine-substituted aryl, or R¹ is a heteroaryl having at leastone nitrogen atom;

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl; and

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments of Formula (I) and Formula (I)(a), R¹ is a group:

In some embodiments of Formula (I) and Formula (I)(a), R¹ is aheteroaryl having at least one nitrogen atom. Examples include, but arenot limited to:

In some embodiments of Formula (I)(a), R¹ is an amine-substituted arylrepresented by Formula (I)(a)(i):

wherein:

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl; and

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments of Formula (I)(a), R¹ is heteroaryl having at leastone nitrogen represented by Formula (I)(a)(ii):

wherein:

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl; and

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments of Formula (I)(a), R¹ is heteroaryl having at leastone nitrogen represented by Formula (I)(a)(iii):

wherein:

n=0 to 10, saturated or unsaturated;

each occurrence of R^(x) and R^(y) is present or absent (depending uponchain saturation), and is each independently H or alkyl; and

R² is selected from the group consisting of: H, alkyl, alkenyl andalkynyl,

or a pharmaceutically acceptable salt or prodrug thereof.

Exemplary compounds of Formula (I)(a) are given below, in which R¹ isheteroaryl, or aryl substituted with amino; n=2, saturated, R^(x) andR^(y) are each H, R² is H, and R³ is substituted cycloalkyl.

Also provided is a pharmaceutically acceptable salt or prodrug thereofof each of the compounds represented by the Formulas described above.Each of the Formulas provided herein may be optionally substituted(e.g., from 1 to 3 or 4 times) with independently selected H, halo,hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy,nitro, carbonyl, carboxy, etc., as desired.

C. COMPOSITIONS

In some embodiments, biofilm inhibiting compositions are provided,comprising a carrier and an effective amount of active compound.“Biofilm” or “biofilms” refer to communities of microorganisms that areattached to a substrate. The microorganisms often excrete a protectiveand adhesive matrix of polymeric compounds. They often have structuralheterogeneity, genetic diversity, and complex community interactions.“Biofilm inhibiting”, “biofilm reducing”, “biofilm resistant”, “biofilmcontrolling” or “antifouling” refer to inhibition of the establishmentor growth of a biofilm, or decrease in the amount of organisms thatattach and/or grow upon a substrate. As used herein, a “substrate” caninclude any living or nonliving structure. For example, biofilms oftengrow on synthetic materials submerged in an aqueous solution or exposedto humid air, but they also can form as floating mats on a liquidsurface, in which case the microorganisms are adhering to each other orto the adhesive matrix characteristic of a biofilm.

An “effective amount” of a biofilm inhibiting composition is that amountwhich is necessary to carry out the composition's function of inhibitinga biofilm.

In some embodiments, the carrier is a pharmaceutically acceptablecarrier. A “pharmaceutically acceptable carrier” as used herein refersto a carrier that, when combined with an active compound of the presentinvention, facilitates the application or administration of that activecompound for its intended purpose to prevent or inhibit biofilmformation, or remove an existing biofilm. The active compounds may beformulated for administration in a pharmaceutically acceptable carrierin accordance with known techniques. See, e.g., Remington, The ScienceAnd Practice of Pharmacy (9^(th) Ed. 1995). The pharmaceuticallyacceptable carrier must, of course, also be acceptable in the sense ofbeing compatible with any other ingredients in the composition. Thecarrier may be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose composition, for example, atablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight ofthe active compound. One or more active compounds may be included in thecompositions of the invention, which may be prepared by any of thewell-known techniques of pharmacy comprising admixing the components,optionally including one or more accessory ingredients.

In general, compositions may be prepared by uniformly and intimatelyadmixing the active compound with a liquid or finely divided solidcarrier, or both, and then, if necessary, shaping the resulting mixture.For example, a tablet may be prepared by compressing or molding a powderor granules containing the active compound, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the compound in a free-flowing form,such as a powder or granules optionally mixed with a binder, lubricant,inert diluent, and/or surface active/dispersing agent(s). Molded tabletsmay be made by molding, in a suitable machine, the powdered compoundmoistened with an inert liquid binder.

The compositions of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound that isbeing used. Routes of parenteral administration include intrathecalinjection and intraventricular injection into a ventricle of the brain.

Compositions suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchcompositions may be prepared by any suitable method of pharmacy, whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above).

Compositions suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavored base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Compositions of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes thatrender the composition isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The compositions may bepresented in unit/dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.

For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising an active compoundas described herein, or a salt or prodrug thereof, in a unit dosage formin a sealed container. The compound or salt is provided in the form of alyophilizate that is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent that is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

Compositions suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by mixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Compositions suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers that may be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Compositions suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Compositionssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3 (6):318(1986)) and typically take the form of an optionally buffered aqueoussolution of the active compound.

Also provided in some embodiments are compositions comprising an activecompound and a biocide. A “biocide” as used herein refers to a substancewith the ability to kill or to inhibit the growth of microorganisms(e.g., bacteria, fungal cells, protozoa, etc.), which substance is notan active compound give above in Section B. Common biocides includeoxidizing and non-oxidizing chemicals. Examples of oxidizing biocidesinclude chlorine, chlorine dioxide, and ozone. Examples of non-oxidizingbiocides include quaternary ammonium compounds, formaldehyde, andanionic and non-anionic surface agents. Chlorine is the most commonbiocide used in sanitizing water systems.

An “antibiotic” as used herein is a type of “biocide.” Commonantibiotics include aminoglycosides, carbacephems (e.g., loracarbef),carbapenems, cephalosporins, glycopeptides (e.g., teicoplanin andvancomycin), macrolides, monobactams (e.g., aztreonam) penicillins,polypeptides (e.g., bacitracin, colistin, polymyxin B), quinolones,sulfonamides, tetracyclines, etc. Antibiotics treat infections by eitherkilling or preventing the growth of microorganisms. Many act to inhibitcell wall synthesis or other vital protein synthesis of themicroorganisms.

Aminoglycosides are commonly used to treat infections caused byGram-negative bacteria such as Escherichia coli and Klebsiella,particularly Pseudomonas aeroginosa. Examples of aminoglycosidesinclude, but are not limited to amikacin, gentamicin, kanamycin,neomycin, netilmicin, streptomycin, tobramycin, and paromomycin.

Carbapenems are broad-spectrum antibiotics, and include, but are notlimited to, ertapenem, doripenem, imipenem/cilstatin, and meropenem.

Cephalosporins include, but are not limited to, cefadroxil, cefazolin,cefalotin (cefalothin), cefalexin, cefaclor, cefamandole, cefoxitin,cefprozil, loracarbef, cefuroxime, cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefepime, cefpirome, and ceftobiprole.

Macrolides include, but are not limited to, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,troleandomycin, telithromycin and spectinomycin.

Penicillins include, but are not limited to, amoxicillin, ampicillin,azlocillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin,flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin,penicillin, piperacillin and ticarcillin.

Quinolones include, but are not limited to, ciprofloxacin, enoxacin,gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin,norfloxacin, ofloxacin and trovafloxacin.

Sulfonamides include, but are not limited to, mafenide, prontosil,sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine,sulfisoxazole, trimethoprim, and co-trimoxazole(trimethoprim-sulfamethoxazole).

Tetracyclines include, but are not limited to, demeclocycline,doxycycline, minocycline, oxytetracycline and tetracycline.

Other antibiotics include arsphenamine, chloramphenicol, clindamycin,lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone,isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin,platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampin(rifampicin), timidazole, etc.

In some embodiments, a dentifrice composition is provided comprising theactive compounds. A “dentifrice” is a substance that is used to cleanthe teeth. It may be in the form of, e.g., a paste or powder. Commonlyknown dentifrices include toothpaste, mouthwash, chewing gum, dentalfloss, and dental cream. Other examples of dentifrices includetoothpowder, mouth detergent, troches, dental or gingival massage cream,dental strips, dental gels, and gargle tablets. Examples of dentifricecompositions comprising toothpaste and mouthwash are found in U.S. Pat.No. 6,861,048 (Yu et al.); U.S. Pat. No. 6,231,836 (Taldkhtalian etal.); and U.S. Pat. No. 6,331,291 (Glace et al.); each incorporated byreference herein in their entirety.

A coating composition is also provided. A “coating” as used herein isgenerally known. Any of a variety of organic and aqueous coatingcompositions, with or without pigments, may be modified to containbiofilm inhibiting compositions as described herein, including but notlimited to those described in U.S. Pat. Nos. 7,109,262, 6,964,989,6,835,459, 6,677,035, 6,528,580, 6,235,812, etc., each incorporated byreference herein in their entirety.

In general, the coatings comprise a film-forming resin, an aqueous ororganic solvent that disperses the resin; and, optionally, at least onepigment. Other ingredients such as colorants, secondary pigments,stabilizers and the like can be included if desired. However, for use inthe present invention the compositions further comprise one or morebiofilm inhibiting compounds as described herein, which may be carriedby or dispersed in the solvent and/or resin, so that the biofilminhibiting compounds are dispersed or distributed on the substrate anarticle coated. A resin may carry the biofilm inhibiting compoundsthrough covalent attachment through means well known in the art. Theresin may comprise, for example, a polymeric material. A polymericmaterial is a material that is comprised of large molecules made fromassociated smaller repeating structural units, often covalently linked.Common examples of polymeric materials are unsaturated polyester resins,and epoxy resins.

Any suitable article can be coated, in whole or in part, with acomposition of the invention. Suitable articles include, but are notlimited to, automobiles and airplanes (including substrates such as wingand propeller surfaces for aerodynamic testing), boat vessel hulls(including interior and exterior surfaces thereof), pressure vessels(including interior and exterior surfaces thereof) medical implants,windmills, etc. Coating of the article with the composition can becarried out by any suitable means, such as by brushing, spraying,electrostatic deposition, dip coating, doctor blading, etc.

D. METHODS OF USE

Methods of controlling biofilm formation on a substrate are disclosed,comprising the step of administering an active compound to a substratein an amount effective to inhibit biofilm formation. A “substrate” asused herein is a base on which an organism, such as those commonly foundin biofilms, may live. The term “substrate,” as used herein, refers toany substrate, whether in an industrial or a medical setting, thatprovides or can provide an interface between an object and a fluid,permitting at least intermittent contact between the object and thefluid. A substrate, as understood herein, further provides a plane whosemechanical structure, without further treatment, is compatible with theadherence of microorganisms. Substrates compatible with biofilmformation may be natural or synthetic, and may be smooth or irregular.Fluids contacting the substrates can be stagnant or flowing, and canflow intermittently or continuously, with laminar or turbulent or mixedflows. A substrate upon which a biofilm forms can be dry at times withsporadic fluid contact, or can have any degree of fluid exposureincluding total immersion. Fluid contact with the substrate can takeplace via aerosols or other means for air-borne fluid transmission.

Biofilm formation with health implications can involve those substratesin all health-related environments, including substrates found inmedical environments and those substrates in industrial or residentialenvironments that are involved in those functions essential to humanwell being, for example, nutrition, sanitation and the prevention ofdisease. Substrates found in medical environments include the inner andouter aspects of various instruments and devices, whether disposable orintended for repeated uses. Examples include the entire spectrum ofarticles adapted for medical use, including scalpels, needles, scissorsand other devices used in invasive surgical, therapeutic or diagnosticprocedures; implantable medical devices, including artificial bloodvessels, catheters and other devices for the removal or delivery offluids to patients, artificial hearts, artificial kidneys, orthopedicpins, plates and implants; catheters and other tubes (includingurological and biliary tubes, endotracheal tubes, peripherablyinsertable central venous catheters, dialysis catheters, long termtunneled central venous catheters, peripheral venous catheters, shortterm central venous catheters, arterial catheters, pulmonary catheters,Swan-Ganz catheters, urinary catheters, peritoneal catheters), urinarydevices (including long term urinary devices, tissue bonding urinarydevices, artificial urinary sphincters, urinary dilators), shunts(including ventricular or arterio-venous shunts); prostheses (includingbreast implants, penile prostheses, vascular grafting prostheses, heartvalves, artificial joints, artificial larynxes, otological implants),vascular catheter ports, wound drain tubes, hydrocephalus shunts,pacemakers and implantable defibrillators, and the like. Other exampleswill be readily apparent to practitioners in these arts. Substratesfound in the medical environment also include the inner and outeraspects of pieces of medical equipment, medical gear worn or carried bypersonnel in the health care setting. Such substrates can includecounter tops and fixtures in areas used for medical procedures or forpreparing medical apparatus, tubes and canisters used in respiratorytreatments, including the administration of oxygen, of solubilized drugsin nebulizers and of anesthetic agents. Also included are thosesubstrates intended as biological barriers to infectious organisms inmedical settings, such as gloves, aprons and faceshields. Commonly usedmaterials for biological barriers may be latex-based or non-latex based.Vinyl is commonly used as a material for non-latex surgical gloves.Other such substrates can include handles and cables for medical ordental equipment not intended to be sterile. Additionally, suchsubstrates can include those non-sterile external substrates of tubesand other apparatus found in areas where blood or body fluids or otherhazardous biomaterials are commonly encountered.

Substrates in contact with liquids are particularly prone to biofilmformation. As an example, those reservoirs and tubes used for deliveringhumidified oxygen to patients can bear biofilms inhabited by infectiousagents. Dental unit waterlines similarly can bear biofilms on theirsubstrates, providing a reservoir for continuing contamination of thesystem of flowing an aerosolized water used in dentistry. Sprays,aerosols and nebulizers are highly effective in disseminating biofilmfragments to a potential host or to another environmental site. It isespecially important to health to prevent biofilm formation on thosesubstrates from where biofilm fragments can be carried away by sprays,aerosols or nebulizers contacting the substrate.

Other substrates related to health include the inner and outer aspectsof those articles involved in water purification, water storage andwater delivery, and articles involved in food processing. Substratesrelated to health can also include the inner and outer aspects of thosehousehold articles involved in providing for nutrition, sanitation ordisease prevention. Examples can include food processing equipment forhome use, materials for infant care, tampons and toilet bowls.“Substrate” as used herein also refers to a living substrate, such asthe inner ear of a patient.

Substrates can be smooth or porous, soft or hard. Substrates can includea drainpipe, glaze ceramic, porcelain, glass, metal, wood, chrome,plastic (e.g., thermoplastic such as polyethylene, polypropylene,polystyrene, polyvinyl chloride or polytetrafluoroethylene (PTFE);thermosetting polymer, etc.) or other polymeric material, vinyl, andlaminates thereof such as Formica® brand laminate, or any other materialthat may regularly come in contact with an aqueous solution in whichbiofilms may form and grow. The substrate can be a substrate commonlyfound on household items such as shower curtains or liners, upholstery,laundry, and carpeting.

A substrate on which biofilm inhibiting is important is that of a shiphull. Biofilms, such as those of Halomonas pacifica, promote thecorrosion of the hull of ships and also increase the roughness of thehull, increasing the drag on the ship and thereby increasing fuel costs.The biofilm can also promote the attachment of larger living structuressuch as barnacles on the ship hull. Fuel can account for half of thecost of marine shipping, and the loss in fuel efficiency due to biofilmformation is substantial.

Substrates on which biofilms can adhere include those of livingorganisms, as in the case of humans with chronic infections caused bybiofilms, as discussed above. Biofilms can also form on the substratesof food contact surfaces, such as those used for processing seafood, andalso on food products themselves. Examples of seafood products that mayhave biofilm contamination include oysters. Human infections caused bythe ingestion of raw oysters has been linked to Vibrio vulnificusbacterium. Vibrio bacteria attach to algae and plankton in the water andtransfer to the oysters and fish that feed on these organisms.

Other examples of substrates or devices on which biofilms can adhere canbe found in U.S. Pat. Nos. 5,814,668 and 7,087,661; and U.S. Pat. Appln.Publication Nos. 2006/0228384 and 2006/0018945, each of which isincorporated herein by reference in its entirety.

In some embodiments, methods of enhancing the effects of a biocide aredisclosed, comprising the step of administering an active compound incombination with a biocide, the active compound being administered in anamount effective to enhance the effects of the biocide.

“Administering” or “administration of” an active compound and/or biocideas used herein in inclusive of contacting, applying, etc. (e.g.,contacting with an aqueous solution, contacting with a surface (e.g., ahospital surface such as a table, instrumentation, etc.)), in additionto providing to a subject (for example, to a human subject in need oftreatment for a microbial infection).

“Enhancing” the effects of a biocide by administering an active compoundin combination with the biocide refers to increasing the effectivenessof the biocide, such that the microorganism killing and/or growthinhibition is higher at a certain concentration of the biocideadministered in combination with the active compound than without. Insome embodiments, a bacteria or other microorganism is “sensitized” tothe effects of a biocide, such that the bacteria or other microorganismthat was resistant to the biocide prior to administering the activecompound (e.g., little to none, or less than 20, 10, 5 or 1% are killedupon application) is rendered vulnerable to that biocide upon or afteradministering the active compound (e.g., greater than 20, 30, 40, 50,60, 70, 80, 90, or 95% or more are killed).

As used herein, the administration of two or more compounds (inclusiveof active compounds and biocides) “in combination” means that the twocompounds are administered closely enough in time that theadministration of or presence of one alters the biological effects ofthe other. The two compounds may be administered simultaneously(concurrently) or sequentially.

Simultaneous administration of the compounds may be carried out bymixing the compounds prior to administration, or by administering thecompounds at the same point in time but at different anatomic sites orusing different routes of administration, or administered at timessufficiently close that the results observed are indistinguishable fromthose achieved when the compounds are administered at the same point intime.

Sequential administration of the compounds may be carried out byadministering, e.g., an active compound at some point in time prior toadministration of a biocide, such that the prior administration ofactive compound enhances the effects of the biocide (e.g., percentage ofmicroorganisms killed and/or slowing the growth of microorganisms). Insome embodiments, an active compound is administered at some point intime prior to the initial administration of a biocide. Alternatively,the biocide may be administered at some point in time prior to theadministration of an active compound, and optionally, administered againat some point in time after the administration of an active compound.

Also disclosed is a method of controlling biofilm formation wherein thebiofilm comprises Gram-negative or Gram-positive bacteria.

“Gram-negative” bacteria are those that do not retain crystal violet dyeafter an alcohol wash in the Gram staining protocol, while“Gram-positive” bacteria are those that are stained dark blue or violetcolor after an alcohol wash in the Gram staining protocol. This is dueto structural properties in the cell walls of the bacteria.Gram-positive bacterial retain the crystal violet color due to a highamount of peptidoglycan in the cell wall.

Many genera and species of Gram-negative and Gram-positive bacteria arepathogenic. A “genus” is a category of biological classification rankingbetween the family and the species, comprising structurally orphylogenetically related species, or an isolated species exhibitingunusual differentiation. It is usually designated by a Latin orlatinized capitalized singular noun. Examples of genera ofbiofilm-forming bacteria affected by active compounds of this inventioninclude, but are not limited to, Pseudomonas, Bordetella, Vibrio,Haemophilus, Halomonas, and Acinetobacter.

“Species” refer to a category of biological classification ranking belowthe genus, and comprise members that are structurally orphylogenetically related, or an isolated member exhibiting unusualdifferentiation. Species are commonly designated by a two-part name,which name includes the capitalized and italicized name of the genus inwhich the species belongs as the first word in the name, followed by thesecond word that more specifically identifies the member of the genus,which is not capitalized. Examples of species of bacteria capable offorming biofilms that are affected by active compounds of the presentinvention include Pseudomonas aeuroginosa, Bordetella bronchiseptica,Bordetella pertussis, Staphylococcus aureus, Vibrio vulnificus,Haemophilus influenzae, Halomonas pacifica, and Acinetobacter baumannii.

Gram-negative bacteria include members of the phylum proteobacteria,which include genus members Escherichia, Salmonella, Vibrio, andHelicobacter.

Other examples of Gram-negative bacteria include, but are not limitedto, bacteria of the genera Klebsiella, Proteus, Neisseria, Helicobacter,Brucella, Legionella, Campylobacter, Francisella, Pasteurella, Yersinia,Bartonella, Bacteroides, Streptobacillus, Spirillum, Moraxella andShigella.

Examples of Gram-positive bacteria include, but are not limited to,bacteria of the genera Listeria, Staphylococcus, Streptococcus,Bacillus, Corynebacterium, Enterococcus, Peptostreptococcus, andClostridium. Examples include, but are not limited to, Listeriamonocytogenes, Staphylococcus aureus (including methicillin-resistant S.aureus, or MSRA), Staphylococcus epidermidis, Streptococcus pyogenes,Streptococcus pneumoniae, Bacillus cereus, Bacillus anthracis,Clostridium botulinum, Clostridium perfringens, Clostridium difficile,Clostridium tetani, Corynebacterium diphtheriae, Corynebacteriumulcerans, Enterococcus faecium (including vancomycin-resistant E.faecium, or VRE), and Peptostreptococcus anaerobius.

Additional bacteria genera in which compounds disclosed herein may beuseful in controlling biofilms include, but are not limited to,Actinomyces, Propionibacterium, Nocardia and Streptomyces. Actinomycesis a Gram-positive genus that includes opportunistic pathogens in humansand animals, e.g., in the oral cavity, and can cause actinomycosis(cause by, e.g., Actinomyces israelii). Propionibacterium acnes is aGram-positive species that can cause acne and chronic blepharitis andendophthalmitis (e.g., after intraocular surgury). Nocardia is aGram-positive genus that includes opportunistic pathogenic speciescausing, e.g., slowly progressive pneumonia, encephalitis, etc.Streptomyces is a Gram-positive genus that occasionally are found inhuman infections, such as mycetoma (caused by, e.g., S. somaliensis andS. sudanensis).

A method for treating a chronic bacterial infection in a subject in needthereof is disclosed, comprising administering active compound to saidsubject in an amount effective to inhibit, reduce, or remove a biofilmcomponent of said chronic bacterial infection. “Treating” as used hereinrefers to any type of activity that imparts a benefit to a patientafflicted with a disease, including improvement in the condition of thepatient (e.g., in one or more symptoms), delay in the progression of thedisease, delay in onset of the disease, etc. The present invention isprimarily concerned with the treatment of human subjects, but theinvention may also be carried out on animal subjects, particularlymammalian subjects (e.g., mice, rats, dogs, cats, rabbits, and horses),avian subjects (e.g., parrots, geese, quail, pheasant), livestock (e.g.,pigs, sheep, goats, cows, chickens, turkey, duck, ostrich, emu), reptileand amphibian subjects, for veterinary purposes or animal husbandry, andfor drug screening and drug development purposes.

A “chronic bacterial infection” is a bacterial infection that is of along duration or frequent recurrence. For example, a chronic middle earinfection, or otitis media, can occur when the Eustachian tube becomesblocked repeatedly due to allergies, multiple infections, ear trauma, orswelling of the adenoids. The definition of “long duration” will dependupon the particular infection. For example, in the case of a chronicmiddle ear infection, it may last for weeks to months. Other knownchronic bacterial infections include urinary tract infection (mostcommonly caused by Escherichia coli and/or Staphylococcussaprophyticus), gastritis (most commonly caused by Helicobacter pylori),respiratory infection (such as those commonly afflicting patents withcystic fibrosis, most commonly caused by Pseudomonas aeuroginosa),cystitis (most commonly caused by Escherichia coli), pyelonephritis(most commonly caused by Proteus species, Escherichia coli and/orPseudomonas species), osteomyelitis (most commonly caused byStaphylococcus aureus, but also by Escherichia coli), bacteremia, skininfection, rosacea, acne, chronic wound infection, infectious kidneystones (can be caused by Proteus mirabilis), bacterial endocarditis, andsinus infection. A common infection afflicting pigs is atrophic rhinitis(caused by Bordatella species, e.g. Bordatella bronchiseptica,Bordatella rhinitis, etc.).

Various nosocomial infections that are especially prevalent in intensivecare units implicate Acinetobacter species such as Acinetobacterbaumannii and Acinetobacter lwoffi. Acinetobacter baumanni is a frequentcause of nosocomial pneumonia, and can also cause skin and woundinfections and bacteremia. Acinetobacter lwoffi causes meningitis. TheAcinetobacter species are resistant to many classes of antibiotics. TheCDC has reported that bloodstream infections implicating Acinetobacterbaumanni were becoming more prevalent among service members injuredduring the military action in Iraq and Afghanistan.

Staphylococcus aureus is a common cause of nosocomial infections, oftencausing post-surgical wound infections. Staphylococcus aureus can alsocause variety of other infections in humans (e.g., skin infections), andcan contribute to mastitis in dairy cows. Staphylococcus aureus hasbecome resistant to many of the commonly used antibiotics.

E. DEVICES

Medical devices comprising a substrate and an effective amount of activecompound are also disclosed. “Medical device” as used herein refers toan object that is inserted or implanted in a subject or applied to asurface of a subject. Common examples of medical devices include stents,fasteners, ports, catheters, scaffolds and grafts. A “medical devicesubstrate” can be made of a variety of biocompatible materials,including, but not limited to, metals, ceramics, polymers, gels, andfluids not normally found within the human body. Examples of polymersuseful in fabricating medical devices include such polymers assilicones, rubbers, latex, plastics, polyanhydrides, polyesters,polyorthoesters, polyamides, polyacrylonitrile, polyurethanes,polyethylene, polytetrafluoroethylene, polyethylenetetraphthalate, etc.Medical devices can also be fabricated using naturally-occurringmaterials or treated with naturally-occurring materials. Medical devicescan include any combination of artificial materials, e.g., combinationsselected because of the particular characteristics of the components.Medical devices can be intended for short-term or long-term residencewhere they are positioned. A hip implant is intended for several decadesof use, for example. By contrast, a tissue expander may only be neededfor a few months, and is removed thereafter.

Some examples of medical devices are found in U.S. Pat. No. 7,081,133(Chinn et al.); U.S. Pat. No. 6,562,295 (Neuberger); and U.S. Pat. No.6,387,363 (Gruskin); each incorporated by reference herein in itsentirety.

F. COVALENT COUPLING OF ACTIVE COMPOUNDS

In some embodiments, active compounds as described herein are covalentlycoupled to substrates. Examples of substrates include solid supports andpolymers. The polymers, typically organic polymers, may be in solidform, liquid form, dispersed or solubilized in a solvent (e.g., to forma coating composition as described above), etc. The solid support mayinclude the substrate examples as described above to be coated with ortreated with active compounds of the invention.

Covalent coupling can be carried out by any suitable technique. Activecompounds of the present invention may be appended to a substrate viaaldehyde condensation, amine bond, amide or peptide bond, carbon-carbonbond, or any suitable technique commonly used in the art. See also U.S.Patent Application Publication No. 2008/0181923 to Melander et al.,which is incorporated by reference herein. A preferred method accordingto some embodiments is amine or amide bond formation. Further examplesand explanations of these types of reactions can be found in U.S. Pat.No. 6,136,157 (Lindeberg et al.) and U.S. Pat. No. 7,115,653 (Baxter etal.), which are each hereby incorporated by reference in their entirety.

Various coupling reactions can be used to covalently link activecompounds of the present invention to a substrate. Examples of couplingreactions that can be used include, but are not limited to, Hiyama,Suzuki, Sonogashira, Heck, Stille, Negishi, Kumada, Wurtz, Ullmann,Cadiot-Chodkiewicz, Buchwald-Hartwig, and Grignard reactions. Forexample, an active compound that is substituted with a halide (e.g.bromo or chloro) can be coupled to a substrate via a Heck reaction.

Some aspects of the present invention are described in more detail inthe following non-limiting examples.

EXAMPLE 1

Synthesis and bacterial biofilm inhibition studies of ethylN-(2-phenethyl) carbamate derivatives. An 88-member library of compoundsbased upon the bacterial metabolite ethyl N-(2-phenethyl) carbamate(2d), isolated from the marine bacteria SCRC3P79 (Cytophaga sp.), wasevaluated. It had been reported that 2d exhibited moderate antibiofilmactivity against the marine α-proteobacteria Rhodospirillum salexigens.Yamada performed preliminary analogue synthesis by varying the aromaticappendage with substituted benzene rings and the ethyl appendage with ahandful of aliphatic subunits. However, none of the analoguesdemonstrated improved activity in comparison to 2d (Yamada et al., Bull.Chem. Soc. Jpn., 1997, 70, 3061).

Ethyl N-(2-phenethyl) carbamate 2d was synthesized from commerciallyavailable materials by routine acylation methodology (ethylchloroformate/TEA in DCM) (Scheme 1). Compound 2d was isolated in 96%yield without recourse to chromatographic purification.

Similar to the results reported in Yamada et al., 2d displayed mediocreantibiofilm activity against R. salexigens, giving a 59.7% inhibition ata 200 μM concentration as judged by a crystal violet reporter assay (seeO'Toole et al., Mol. Microbiol., 1998, 30, 295).

Interestingly, we found that a 200 μM concentration of 2d also displayedactivities against various medically relevant bacterial strains,inhibiting 63.1%, 68.1%, 80.2%, 52.0% and 40.8% of biofilm formation forS. epidermidis, methicillin-resistant S. aureus (MRSA),vancomycin-resistant Enterococcus faecium (VRE), multi-drug resistantAcinetobacter baumannii (MDRAB), and E. coli respectively (Table 1).

TABLE 1 Biofilm inhibition activity of 2d against various bacteria.Strain % Inhibition (200 μM 2d) S. epidermidis 63.1 MRSA 68.1 VRE 80.2R. salexigens 59.7 MDRAB 52.0 E. coli 40.8

After successfully obtaining antibiofilm activity for 2d againstmedically relevant bacteria, a three-part structure-activity analysiswas designed (see Scheme 1), which entailed a systematic modulation ofthe metabolite's aromatic head region, carbamate linkage, and tailgroup.

These natural product analogues were synthesized using the same methodused to prepare 2d. Specifically, the respective amine was reacted with0.9 equivalent of the requisite chloroformate, isocyanate, dicarbonate,or isothiocyanate in the presence of 2.0 equivalents of triethylamine indichloromethane. Each of the listed amines was reacted independentlywith each acylating reagent to produce the 88-member library in yieldsranging from 76-98%.

Various aromatic head groups were used, incorporating the indole,triazole, indane, tetrahydroquinoline, indoline, and pyridine, as wellas para-amino, para-methoxy, and para-bromo substituted phenyl rings.The carbamate heteroatomic core was varied through the substitution witha thiocarbamate, urea, and thiourea linkages. Tail modifications weremade through the incorporation of the (−)-menthyl, benzyl, t-butyl andcholesteryl groups (Scheme 2).

Once 2a-12h had been synthesized, they were screened for their abilityto inhibit biofilm formation of S. epidermidis, MRSA, VRE, R.salexigens, MDRAB and E. coli at a 200 μM concentration. None of thecompounds displayed notable antibiofilm activity against S. epidermidis,VRE, R. salexigens, or MDRAB. However, compounds 4c, 8a, 9a, and 10adisplayed greater than 90% inhibition of MRSA biofilms at 200 μMconcentration. Furthermore, compounds 5a, 9a, and 10c exhibited greaterthan 80% inhibition of E. coli biofilms at 200 μM concentration.

Dose-response curves were generated for the lead compounds for theinhibition of MRSA and E. coli biofilms (Table 2). Non-bactericidalantibiofilm activity was verified through colony count analysis of theplanktonic viability in the presence and in the absence of each compoundat their IC₅₀ value (i.e. the concentration that inhibits 50% of biofilmformation). Against MRSA, IC₅₀ values were determined to be 49.8 μM,4.87 μM and 4.70 μM for 8a, 9a, and 10a respectively. Against E. coli,IC₅₀ values were determined to be 28.3 μM, 34.6 μM and 66.4 μM for 5a,9a, and 10c, respectively.

TABLE 2 Biofilm inhibition (IC₅₀) against MRSA and E. coli. MRSA IC₅₀ E.coli IC₅₀ Compound (μM) (μM) 5a — 28.3 8a 49.8 — 9a 4.87 34.6 10a  4.70— 10c  — 66.4

Given the potency of our lead compounds toward inhibiting MRSA biofilms,we next explored their activity against various other S. aureus strains.Specifically, 8a, 9a, and 10a were screened against three additional S.aureus strains (ATCC #'s 29213, 29740, and 25923). IC₅₀ values weredetermined for each compound against each of the S. aureus strains. Insome cases, the compounds were found to be more potent than they wereagainst MRSA.

IC₅₀ values for compound 8a were found to be 21.2 μM, 24.3 μM and 71.9μM against 29213, 29740 and 25923 respectively. For 9a they were foundto be 124 μM, 82.2 μM and 19.7 μM for 29213, 29740 and 25923respectively. Lastly, for compound 10a, which was found to be the mostactive compound overall, IC₅₀ values were determined to be 4.70 μM, 2.84μM and 37.4 μM for 29213, 29740 and 25923 respectively (Table 3). Again,planktonic viability in the presence of the test compounds was verifiedthrough colony count analysis.

TABLE 3 Biofilm inhibition (IC₅₀) against other S. aureus strains. 29213IC₅₀ 29740 IC₅₀ 25923 IC₅₀ Compoud (μM) (μM) (μM) 8a 21.2 24.3 71.9 9a124 82.2 19.7 10a  4.70 2.85 37.4

Interestingly, our most potent inhibitors of the S. aureus strainsincluding MRSA contained (−)-menthyl carbamates. Indeed, (−)-menthol andits derivatives have long been shown to have various antimicrobial andantiplasmid effects on bacteria (Schelz et al., Fitoterapia, 2006, 77,279; Filoche et al., Oral Microb. Immun., 2005, 20, 221; Iscan et al.,J. Agric. Food Chem., 2002, 50, 3943; Kurita et al. Agric. Biol. Chem.,1982, 46, 159; Aridogan et al., Arch. Pharm. Res. Vol. 2002, 25, 860.).Along with (−)-menthol (13), the related natural products thymol (14)and carvacrol (15) (Scheme 3, dashed box) are also known to possessantimicrobial activity (Arfa et al., Lett. Appl. Microbiol., 2006, 43,149; Sivropoulou et al., J. Agric. Food Chem., 1996, 44, 1202; Ultee etal., J. Food Protect., 2000, 63, 620). In light of this observation, weprepared the thymyl and carvacryl carbamate analogues of 9a and 10a. Wechose these two compounds for analogue design because they had thelowest IC₅₀ values against MRSA and both worked well against 29213,29740, and 25923 (see Tables 2 and 3). Additionally, we prepared thestereochemical antipodes of 9a and 10a by employing (+)-menthylcarbamate. Finally, we prepared the cyclohexyl carbamate derivatives of9a and 10a as a control (Scheme 3).

With compounds 91-101 in hand, they were then screened for biofilminhibition activity along with (−)-menthol and (−)-menthol methyl etheragainst MRSA and 29213. Interestingly, none of the analogues depicted inScheme 3 displayed any notable biofilm inhibition activity againsteither MRSA or 29213 at a 200 μM concentration, with the exception of 9kand 10k. These compounds were found to have identical antibiofilmproperties as their enantiomers, 9a and 10a. Importantly, both(−)-menthol and (−)-menthol methyl ether were found to be completelyinactive.

Lastly, 2d, 8a, 9a, and 10a were preliminarily screened forcytotoxicity. This was assessed using a red blood cell hemolysis assayusing difibrinated sheep blood. In each case, the carbamates were foundto show no red blood cell lysis up to the highest concentration tested(1.2 mM).

In summary, by targeting analogues of the bacterial metabolite 2d, wehave discovered a novel class of biofilm inhibitors based upon a menthylcarbamate scaffold. Two potent compounds (9a and 10a) display lowmicromolar IC₅₀ values for the inhibition of various S. aureus biofilmsincluding those from the medically relevant MRSA. This scaffoldrepresents a unique class of compounds for combating bacterial biofilms.

GENERAL EXPERIMENTAL

All reagents used for chemical synthesis were purchased fromcommercially available sources and used without further purification.Chromatography was performed using 60 {acute over (Å)} mesh standardgrade silica gel from Sorbtech. NMR solvents were obtained fromCambridge Isotope Labs and used as is. ¹H NMR (300 MHz or 400 MHz) and¹³C NMR (75 MHz or 100 MHz) spectra were recorded at 25° C. on VarianMercury spectrometers. Chemical shifts (8) are given in ppm relative totetramethylsilane or the respective NMR solvent; coupling constants (J)are in hertz (Hz). Abbreviations used are s=singlet, bs=broad singlet,d=doublet, dd=doublet of doublets, t=triplet, dt=doublet of triplets,bt=broad triplet, qt=quartet, m=multiplet, bm=broad multiplet andbr=broad. Mass spectra were obtained at the NCSU Department of ChemistryMass Spectrometry Facility.

General Procedure for Compounds 2a-2h, 3a-3h, 4a-4-h, 5a-5h, 6a-6h,7a-7h, 11a-h and 12a-12h.

Ten mL of dichloromethane and a stir bar was added to 100-200 mg of theamine. Two equivalents of triethylamine was then added and the reactionmixture was cooled to 0° C. while stirring. Then, 0.9 equivalents of thechloroformate, isocyanate or thioisocyanate was added dropwise to thereaction mixture and was allowed to slowly warm to room temperature andcontinued stirring overnight. The reaction mixture was then diluted withmore dichloromethane, washed twice with 1N HCl, washed twice with brine,dried with sodium sulfate and then concentrated in vacuo.

General Procedure for Compounds 8a-8h, 9a-9l and 10a-10l.

Ten mL of dichloromethane and a stir bar was added to 100-200 mg of theamine. Two equivalents of triethylamine was then added and the reactionmixture was cooled to 0° C. while stirring. Then, 0.9 equivalents of thechloroformate, isocyanate or thioisocyanate was added dropwise to thereaction mixture and was allowed to slowly warm to room temperature andcontinued stirring overnight. The reaction mixture was then diluted withmore dichloromethane, washed twice with brine, dried with sodium sulfateand then concentrated in vacuo. The crude mixture was then purified viaflash chromatography on silica gel using a 2.5%-10%methanol/dichlormethane eluent.

2-(2H-1,2,3-triazol-2-yl)ethanamine (5)

White solid, mp=129-131° C. ¹H NMR (300 MHz, CDCl₃) δ 7.43 (s, 2H), δ4.55 (t, J=1.2 Hz, 2H), δ 3.33 (t, J=5.7 Hz, 2H) ppm; ¹³C NMR (75 MHz,CDCl₃) δ 135.1, 51.3, 38.8 ppm; IR ν_(max) (cm⁻¹) 3054, 2987, 2306,1421, 1258; HRMS (ESI) calcd for C₄H₈N₄ (M+) 113.0822. found 113.0819.

tert-butyl 2-(2H-1,2,3-triazol-2-yl)ethylcarbamate (5e)

White solid. mp=64-66° C. ¹H NMR (300 MHz, CDCl₃) δ 7.55 (s, 2H), δ 5.15(s, 1H), δ 4.49 (t, J=5.7 Hz, 2H), δ 3.63 (q, J=5.7, 5.1 Hz, 2H), δ 1.36(s, 9H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 155.9, 134.5, 79.8, 54.8, 40.2,28.5, 27.8 ppm; IR ν_(max) (cm⁻¹) 3054, 2986, 1713, 1506; HRMS (ESI)calcd for C₉H₁₆N₄O₂ (M+) 235.1165. found 235.1169.

tert-butyl 2,3-dihydro-1H-inden-2-ylcarbamate (3e)

White solid. mp=51-53° C. ¹H NMR (300 MHz, CDCl₃) δ 7.19 (m, 4H), δ 4.84(s, 1H), δ 4.45 (s, 1H), δ 3.29 (d, J=7.2 Hz, 1H), δ 3.24 (d, J=6.9 Hz,1H), δ 2.80 (d, J=4.8 Hz, 1H), δ 2.75 (d, J=4.8 Hz, 1H), δ 1.43 (s, 9H)ppm; ¹³C NMR (75 MHz, CDCl₃) δ153.5, 140.9, 126.5, 124.7, 79.3, 51.9,40.3, 28.4 ppm; IR ν_(max) (cm⁻¹) 3419, 2321, 1641; HRMS (ESI) calcd forC₁₇H₁₇NO₂ (M+) 256.1308. found 256.1308.

benzyl 2,3-dihydro-1H-inden-2-ylcarbamate (3c)

Light yellow solid. mp=159-161° C. ¹H NMR (300 MHz, CDCl₃) δ 7.35 (s,4H), δ 7.13 (t, J=1.2 Hz, 5H), δ 5.09 (s, 2H), δ 4.56 (s, 2H), δ 3.28(m, 2H), δ 2.77 (m, 2H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 141.3, 136.7,128.8, 126.9, 125.0, 66.9, 51.8, 40.8 ppm; IR ν_(max) (cm⁻¹) 3419, 2977,1691, 1643, 1265; HRMS (ESI) calcd for C₁₇H₁₇NO₂ (M+) 268.1332. found268.1337.

benzyl 2-(pyridin-2-yl)ethylcarbamate (9c)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 8.43 (d, J=4.8 Hz, 1H), δ 7.52(t, J=1.8 Hz, 1H), δ 7.23 (s, 5H), δ 7.06 (m, 2H), δ 5.91 (s, 1H), δ5.04 (s, 2H), δ 3.57 (q, J=6.3 Hz, 6.3 Hz, 2H), δ 2.93 (t, J=6.6 Hz, 2H)ppm; ¹³C NMR (75 MHz, CDCl₃) δ 156.7, 149.4, 137.0, 128.6, 127.1, 123.7,121.7, 66.7, 40.6, 37.8 ppm; 3440, 2092, 1644, 1261; HRMS (ESI) calcdfor C₁₅H₁₆N₂O₂ (M+) 257.1285. found 257.1288.

benzyl 4-aminophenethylcarbamate (8c)

Light yellow solid. mp=70-73° C. ¹H NMR (300 MHz, CDCl₃) δ 7.31 (s, 5H),δ 6.93 (d, J=8.1 Hz, 2H), δ 6.58 (d, J=8.4 Hz, 2H), δ 5.06 (s, 2H), δ4.91 (s, 1H), δ 3.57 (s, 2H), δ 3.36 (q, J=6.6, 6.6 Hz, 2H), δ 2.65 (t,J=6.9 Hz, 2H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 156.6, 145.2, 136.9, 129.8,128.7, 115.6, 66.8, 58.8, 42.7, 35.4 ppm; IR ν_(max) (cm⁻¹) 3389, 1682,1543; HRMS (ESI) calcd for C₁₆H₁₈N₂O₂ (M+) 271.1441. found 271.1446.

benzyl 2-(2H-1,2,3-triazol-2-yl)ethylcarbamate (5c)

Light yellow residue. ¹H NMR (300 MHz, CDCl₃) δ 7.58 (s, 2H), δ 7.28 (m,5H), δ 5.38 (s, 1H), δ 5.15 (s, 2H), δ 4.51 (t, J=5.4 Hz, 2H), δ 3.75(t, 6.3 Hz, 2H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 156.5, 136.5, 134.5,128.4, 67.1, 54.6, 40.6 ppm; IR ν_(max) (cm⁻¹) 3440, 3054, 2986, 2305,1719; HRMS (ESI) calcd for C₁₂H₁₄N₄O₂ (M+) 269.1009. found 269.1011.

ethyl 4-aminophenethylcarbamate (8d)

Light yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 6.95 (d, J=8.1 Hz, 2H), δ6.61 (d, J=6.3 Hz, 2H), δ 4.89 (s, 1H), δ 4.08 (q, J=6.9, 7.2 Hz, 2H), δ3.74 (s, 2H), δ 3.33 (t, J=6.3 Hz, 2H), δ 2.65 (t, J=7.2 Hz, 2H), δ 1.19(t, J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 156.9, 145.0, 129.8,115.7, 60.9, 42.6, 35.4, 14.9 ppm; IR ν_(max) (cm⁻¹) 3346, 2932, 1698,1627; HRMS (ESI) calcd for C₁₁H₁₆N₂O₂ (M+) 209.1285. found 209.1278.

ethyl 2-(pyridin-2-yl)ethylcarbamate (9d)

Light yellow solid. mp=56-58° C. ¹H NMR (300 MHz, CDCl₃) δ 8.41 (d,J=4.8 Hz, 1H), δ 7.49 (t, J=5.7 Hz, 1H), δ 7.02 (m, 2H), δ 5.77 (s, 1H),δ 4.00 (q, J=6.9, 7.2 Hz, 2H), δ 3.50 (q, J=6.3, 6.6 Hz, 2H), δ 2.89 (t,J=6.6 Hz, 2H), δ 1.11 (t, J=6.9 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ159.5, 156.9, 149.3, 136.6, 123.5, 121.6, 60.6, 40.4, 37.9, 14.8 ppm; IRν_(max) (cm⁻¹) 3435, 2091, 1641, 1259; HRMS (ESI) calcd for C₁₄H₁₇N₅O₂S(M+). found.

ethyl 4-bromophenethylcarbamate (11d)

White solid. mp=63-65° C. ¹H NMR (300 MHz, CDCl₃) δ 7.42 (d, J=2.7 Hz,2H), δ 7.07 (d, J=8.7 Hz, 2H), δ 4.94 (s, 1H), δ 4.09 (q, J=6.9, 7.2 Hz,2H), δ 3.39 (q, J=6.6, 6.9 Hz, 2H), δ 2.75 (t, 6.9 Hz, 2H), δ 1.21 (t,J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 156.8, 138.1, 131.7, 120.5,61.0, 42.1, 35.8, 14.9 ppm; IR ν_(max) (cm⁻¹) 3348, 2975, 1691, 1537,1260; HRMS (ESI) calcd for C₁₁H₁₄BrNO₂ (M+) 227.0281. found 227.0279.

ethyl 2-(2H-1,2,3-triazol-2-yl)ethylcarbamate (5d)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.62 (s, 2H), δ 6.31 (s, 1H), δ4.671 (t, J=4.2 Hz, 2H), δ 4.17 (q, J=4.5, 4.5 Hz, 2H), δ 3.31 (s, 2H),δ 1.21 (t, J=6.9 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 202.1, 134.7,54.2, 44.3, 41.8, 14.1 ppm; IR ν_(max) (cm⁻¹) 3434, 1642; HRMS (ESI)calcd for C₁₄H₁₇N₅O₂S (M+). found.

S-ethyl 3-phenylpropylcarbamothioate (4b)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.25-7.10 (m, 5H), δ 6.24 (s,1H), δ 3.26 (t, J=5.7 Hz, 2H), δ 2.82 (q, J=4.8, 6.9 Hz, 2H), δ 2.58 (t,J=7.8 Hz, 2H), δ 1.76 (m, 2H), δ 1.26 (t, J=7.5 Hz, 3H) ppm; ¹³C NMR (75MHz, CDCl₃) δ 137.8, 141.7, 128.7, 126.5, 41.3, 33.4, 32.0, 24.5, 16.1ppm; IR ν_(max) (cm⁻¹) 3318, 3027, 2929, 2867, 1650, 1624, 1215, 700;HRMS (ESI) calcd for C₁₂H₁₇NOS (M+) 224.1104. found 224.1099.

S-ethyl 2-(pyridin-2-yl)ethylcarbamothioate (9b)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 8.37 (d, J=3 Hz, 1H), δ 7.47(t, J=6.0 Hz, 1H), δ 7.01 (m, 2H), δ 6.88 (s, 1H), δ 3.58 (q, J=4.8,4.5, 2H), δ 2.89 (t, J=5.1 Hz, 2H), δ 2.78 (q, J=5.4, 5.4 Hz, 2H), δ1.15 (t, J=5.1 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.3, 149.3,136.8, 123.6, 121.8, 40.7, 37.4, 24.6, 15.9 ppm; IR ν_(max) (cm⁻¹) 3432,2089, 1645, 1213; HRMS (ESI) calcd for C₁₄H₁₇N₅O₂S (M+). found.

S-ethyl indoline-1-carbothioate (6b)

Light yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.97 (d, J=5.7 Hz, 1H), δ7.08-6.88 (m, 4H), δ 3.80 (t, J=7.8 Hz, 2H), δ 2.94 (q, J=6.0, 5.4 Hz,2H), δ 2.89 (t, J=3.3 Hz, 2H), δ 1.31 (t, J=5.7 Hz, 3H) ppm; ¹³C NMR (75MHz, CDCl₃) δ 165.6, 143.0, 131.3, 127.6, 124.9, 123.6, 116.0, 47.3,28.0, 24.7, 15.7 ppm; IR ν_(max) (cm⁻¹) 3053, 2932, 2253, 1712, 1598,1650; HRMS (ESI) calcd for C₁₁H₁₃NOS (M+) 208.0791. found 208.0788.

S-ethyl 4-methoxyphenethylcarbamothioate (12b)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.09 (d, J=8.4 Hz, 2H), δ 6.83(d, J=8.7, 2H), δ 5.96 (s, 1H), δ 3.50 (s, 3H), δ 3.45 (q, J=6.3, 6.6Hz, 2H), δ 2.91 (q, J=3.3, 3.3 Hz, 2H), δ 2.75 (t, J=7.5 Hz, 2H), δ 1.28(t, J=6.9 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 167.7, 158.5, 130.9,129.9, 114.2, 55.4, 43.0, 35.2, 24.4, 16.0 ppm; IR ν_(max) (cm⁻¹) 3323,3034, 2962, 1640, 1514; HRMS (ESI) calcd for C₁₄H₁₇N₅O₂S (M+). found.

S-ethyl 4-aminophenethylcarbamothioate (8b)

Light yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 6.92 (d, J=6.8, 2H), δ 6.59(t, J=6.4 Hz, 2H), δ 5.87 (s, 1H), δ 3.77 (s, 1H), δ 3.41 (t, J=5.6 Hz,2H), δ 2.86 (q, J=7.2, 7.2 Hz, 2H), δ 2.67 (t, J=6.4, 2H), δ 1.25 (t,J=5.2 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 167.6, 145.1, 129.8,115.7, 53.8, 43.0, 35.2, 24.5, 16.0 ppm; IR ν_(max) (cm⁻¹) 3434, 2929,2086, 1647, 1517, 1263, 1219, 970; HRMS (ESI) calcd for C₁₁H₁₆N₂OS (M+)225.1056. found 225.1060.

S-ethyl 2,3-dihydro-1H-inden-2-ylcarbamothioate (3b)

White solid. mp=107-110° C. ¹H NMR (300 MHz, CDCl₃) δ 7.23-7.15 (m, 4H),δ 6.08 (d, J=5.4 Hz, 1H), δ 4.70 (s, 1H), δ 3.26 (dd, J=7.2, 7.2 Hz,2H), δ 2.86 (q, J=5.4, 6.6 Hz, 2H), δ 1.29 (t, J=7.5 Hz, 3H) ppm; ¹³CNMR (75 MHz, CDCl₃) δ 167.4, 141.0, 127.1, 125.0, 52.7, 40.2, 24.5, 16.0ppm; IR ν_(max) (cm⁻¹) 3258, 3019, 2945, 1628; HRMS (ESI) calcd forC₁₂H₁₅NOS (M+) 222.0947. found 222.0944.

S-ethyl 2-(1H-indol-3-yl)ethylcarbamothioate (10b)

Light yellow solid. mp=68-70° C. ¹H NMR (300 MHz, CDCl₃) δ 8.52 (s, 1H),δ 7.54 (d, J=7.8, 1H), δ 7.24 (d, J=8.1, 1H), δ 7.17-7.04 (m, 2H), δ6.82 (d, J=2.1 Hz, 1H), δ 5.74 (s, 1H), δ 3.51 (t, J=6.3 Hz, 2H), δ2.91-2.83 (m, 4H), δ 1.26 (t, J=4.5 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃)δ 168.2, 136.8, 127.6, 122.8, 119.7, 112.6, 42.1, 25.8, 24.7, 16.2 ppm;IR ν_(max) (cm⁻¹) 3418, 3055, 2932, 1657, 1496; HRMS (ESI) calcd forC₁₃H₁₆N₂OS (M+) 249.1056. found 249.1052.

S-ethyl 2-(2H-1,2,3-triazol-2-yl)ethylcarbamothioate (5b)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.59 (s, 1H), δ 6.14 (s, 1H), δ4.56 (t, J=5.1 Hz, 2H), δ 3.83 (q, J=5.7, 5.7 Hz, 2H), δ 2.88 (q, J=7.5,7.5 Hz, 2H), δ 1.25 (t, J=7.5 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ202.1, 134.7, 54.3, 40.5, 24.6, 15.8 ppm; IR ν_(max) (cm⁻¹) 3434, 1647,671; HRMS (ESI) calcd for C₁₄H₁₇N₅O₂S (M+). found.

S-ethyl phenethylcarbamothioate (2b)

White solid. mp=95-98° C. ¹H NMR (300 MHz, CDCl₃) δ 7.30 (m, 5H), δ 6.02(s, 1H), δ 3.53 (q, J=6.6, 6.6 Hz, 2H), δ 2.89 (m, 4H), δ 1.30 (t, J=6.6Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 167.8, 139.0, 129.1, 126.8, 42.9,36.2, 24.5, 16.1 ppm; IR ν_(max) (cm⁻¹) 3399, 2968, 2929, 2870, 2088,1650, 1498; HRMS (ESI) calcd for C₁₁H₁₅NOS (M+) 210.0947. found210.0944.

1-(2-(1H-indol-3-yl)ethyl)-3-ethylthiourea (10h)

Light yellow residue. ¹H NMR (300 MHz, CDCl₃) δ 8.67 (s, 1H), δ 7.58 (d,J=7.2 Hz, 1H), δ 7.33 (d, J=8.1 Hz, 1H), δ 7.18 (t, J=6.9 Hz, 1H), δ7.08 (t, J=7.2 Hz, 1H), δ 6.86 (s, 1H), δ 6.24 (s, 2H), δ 3.69-2.94 (m,6H), δ 1.00 (t, J=6.9 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 180.9,136.7, 127.4, 122.4, 118.9, 112.3, 45.0, 39.4, 25.3, 14.4 ppm; IRν_(max) (cm⁻¹) 3418, 2975, 1634, 1569, 1265, 738, 702; HRMS (ESI) calcdfor C₁₃H₁₇N₃S (M+) 248.1216. found 248.1215.

1-(2-(1H-indol-3-yl)ethyl)-3-ethylurea (10g)

White solid. mp=109-112° C. ¹H NMR (300 MHz, CDCl₃) δ 8.55 (s, 1H), δ7.57 (d, J=7.8 Hz, 1H), δ 7.32-7.05 (m, 3H), δ 6.86 (s, 1H), δ 6.13 (s,2H), δ 3.69 (m, 2H), δ 3.20 (m, 2H), δ 2.94 (t, J=6.3 Hz, 2H), δ 0.97(t, J=7.2 Hz, 3H) 181.0, 136.6, 127.4, 122.9, 119.7, 112.3, 44.6, 39.0,25.2, 14.3 ppm; ¹³C NMR (75 MHz, CDCl₃) δ 180.9, 136.6, 127.4, 122.9,122.4, 119.7, 112.3, 111.9, 44.6, 38.9, 25.2, 14.4 ppm; IR ν_(max)(cm⁻¹) 3944, 3467, 3054, 2986, 2306, 1536; HRMS (ESI) calcd forC₁₃H₁₇N₃O (M+) 232.1444. found 232.1445.

1-(2-(2H-1,2,3-triazol-2-yl)ethyl)-3-ethylthiourea (5h)

White solid. mp=91-93° C. ¹H NMR (300 MHz, CDCl₃) δ 7.57 (d, J=9.6 Hz2H), δ 6.64 (s, 2H), δ 4.62 (q, J=5.1, 3.9 Hz, 2H), δ 4.10 (q, J=5.7,5.4 Hz), δ 3.28 (t, J=5.7 Hz, 2H), δ 1.15 (t, J=9.3 Hz, 3H) ppm; ¹³C NMR(75 MHz, CDCl₃) δ 197.1, 134.7, 100.4, 54.1, 44.2, 14.1 ppm; IR ν_(max)(cm⁻¹) 3419, 1640, 1551; HRMS (ESI) calcd for C₁₄H₁₇N₅O₂S (M+). found.

N-ethylindoline-1-carbothioamide (6h)

Yellow solid. mp=88-91° C. ¹H NMR (300 MHz, CDCl₃) δ 7.83 (d, J=7.8 Hz,2H), δ 7.08-7.01 (m, 2H), δ 6.85 (t, J=7.2 Hz, 1H), δ 6.21 (t, J=4.5 Hz,1H), δ 4.09 (t, J=8.4 Hz, 2H), δ 3.63 (m, 2H), δ 2.87 (t, J=8.4 Hz, 2H),δ 1.18 (t, J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 178.9, 142.5,134.0, 127.1, 125.9, 123.4, 114.9, 53.4, 40.3, 27.3, 14.6 ppm; IRν_(max) (cm⁻¹) 3390, 3279, 3030, 2972, 1638, 1522; HRMS (ESI) calcd forC₁₁H₁₄N₂S (M+) 207.0950. found 207.0946.

1-ethyl-3-(2-(pyridin-2-yl)ethyl)thiourea (9h)

Light yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 8.25 (d, J=4.2 Hz, 1H), δ7.43 (t, J=7.5 Hz, 1H), δ 7.33 (s, 1H), δ 7.01-6.92 (m, 3H), δ 3.70 (t,J=3.9 Hz, 2H), δ 3.21 (m, 2H), δ 2.85 (t, J=6.3 Hz, 2H), δ 0.98 (t,J=6.9 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 202.3, 181.0, 159.4, 150.0,148.9, 137.1, 123.8, 121.9, 43.8, 39.0, 36.8, 24.7, 14.3 ppm; IR ν_(max)(cm⁻¹) 3396, 2974, 2100, 1641, 1556; HRMS (ESI) calcd for C₁₄H₁₇N₅O₂S(M+). found.

1-ethyl-3-(3-phenylpropyl)thiourea (4h)

White solid. mp=51-53° C. ¹H NMR (300 MHz, CDCl₃) δ 7.25-7.11 (m, 5H), δ6.42 (s, 2H), δ 3.42 (m, 2H), δ 3.34 (m, 2H), δ 2.61 (t, J=7.5 Hz, 2H),δ 1.86 (m, 2H), 1.10 (t, J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ181.3, 141.4, 128.7, 126.3, 39.3, 33.9, 30.8, 14.6 ppm; IR ν_(max)(cm⁻¹) 3421, 3276, 3053, 2939, 2865, 1547, 1495; HRMS (ESI) calcd forC₁₂H₁₈N₂S (M+) 223.1263. found 223.1261.

N-ethyl-3,4-dihydroquinoline-1(2H)-carbothioamide (7h)

Light yellow solid. mp=55-57° C. ¹H NMR (300 MHz, CDCl₃) δ 7.09-6.97 (m,4H), δ 6.22 (s, 1H), δ 4.09 (t, J=6.3 Hz, 2H), δ 3.50 (m, 2H), δ 2.59(t, J=6.9 Hz, 2H), δ 1.84 (m, 2H), δ 1.02 (t, J=3.9 Hz, 3H) ppm; ¹³C NMR(75 MHz, CDCl₃) δ 181.9, 138.8, 134.0, 130.3, 126.8, 123.7, 49.1, 40.9,26.7, 24.0, 14.3 ppm; IR ν_(max) (cm⁻¹) 3396, 3034, 2934, 2875, 2211,1640, 1516; HRMS (ESI) calcd for C₁₂H₁₆N₂S (M+) 221.1107. found221.1105.

1-(4-aminophenethyl)-3-ethylthiourea (8h)

Light yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 6.94 (d, J=8.1, 2H), δ 6.57(d, J=4.5 Hz, 2H), δ 6.06 (d, J=10.2 Hz, 2H), δ 3.60 (m, 4H), δ 3.26 (s,2H), δ 2.72 (t, J=6.9 Hz, 2H), δ 1.08 (t, J=7.2 Hz, 3H) ppm; ¹³C NMR (75MHz, CDCl₃) δ 181.3, 145.3, 129.8, 128.3, 115.7, 46.1, 39.1, 34.6, 14.4ppm; IR ν_(max) (cm⁻¹) 3408, 1626, 1553; HRMS (ESI) calcd for C₁₁H₁₇N₃S(M+) 224.1216. found 224.1212.

1-ethyl-3-phenethylthiourea (2h)

White solid. mp=56-58° C. ¹H NMR (300 MHz, CDCl₃) δ 7.27-7.14 (m, 5H), δ6.40 (d, J=10.2 Hz, 2H), δ 3.68 (t, J=5.1 Hz, 2H), δ 3.30 (m, 2H), δ2.84 (t, J=7.2 Hz, 2H), δ 1.08 (t, J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz,CDCl₃) δ 181.4, 138.7, 129.0, 126.9, 45.9, 39.1, 35.6, 14.4 ppm; IRν_(max) (cm⁻¹) 3420, 3269, 3054, 2984, 2935, 2875, 2685, 2306, 2253,1711, 1546; HRMS (ESI) calcd for C₁₁H₁₆N₂S (M+) 209.1107. found209.1103.

1-(2,3-dihydro-1H-inden-2-yl)-3-ethylthiourea (3h)

Grey solid. mp=87-91° C. ¹H NMR (300 MHz, CDCl₃) δ 7.14 (m, 4H), δ 6.62(s, 2H), δ 4.85 (m, 1H), δ 3.50-3.21 (m, 4H), δ 2.83 (dd, J=5.4, 5.1 Hz,2H), δ 1.10 (t, J=7.5 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 181.0,140.8, 127.1, 125.0, 100.4, 55.5, 40.0, 14.6 ppm; IR ν_(max) (cm⁻¹)3267, 3066, 2972, 1673, 1483, 1548; HRMS (ESI) calcd for C₁₂H₁₆N₂S (M+)221.1107. found 221.1106.

1-(4-bromophenethyl)-3-ethylurea (11g)

White solid. mp=137-139° C. ¹H NMR (300 MHz, CDCl₃) δ 7.37 (d, J=8.1 Hz,2H), δ 7.02 (d, J=8.1 Hz, 2H), δ 5.46 (d, J=16.5 Hz, 2H), δ 3.30 (m,2H), δ 3.08 (q, J=6.0, 6.9 Hz, 2H), δ 2.67 (t, J=6.9 Hz, 2H), δ 1.05 (t,J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.1, 138.6, 131.7, 130.7,120.3, 41.6, 36.3, 35.2, 15.8 ppm; IR ν_(max) (cm⁻¹) 3327, 2971, 1620,1488; HRMS (ESI) calcd for C₁₁H₁₅BrN₂O (M+) 271.0441. found 271.0439.

1-(2,3-dihydro-1H-inden-2-yl)-3-ethylurea (3g)

White solid. mp=117° C. ¹H NMR (300 MHz, CDCl₃) δ 7.14 (m, 4H), δ 5.98(s, 2H), δ 4.43 (m, 1H), δ 3.22-3.07 (m, 4H), δ 2.77 (dd, J=6.3, 5.7 Hz,2H), δ 1.07 (t, J=6.9 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.3,141.4, 126.8, 124.9, 51.5, 40.7, 35.1, 15.9 ppm; IR ν_(max) (cm⁻¹) 3348,2968, 1623, 1579, 1259, 736; HRMS (ESI) calcd for C₁₂H₁₆N₂O (M+)205.1335. found 205.1333.

1-(2-(2H-1,2,3-triazol-2-yl)ethyl)-3-ethylurea (5g)

White solid. mp=109° C. ¹H NMR (300 MHz, CDCl₃) δ 7.57 (s, 2H), δ 6.63(d, J=25.5 Hz, 2H), δ 4.62 (m, 2H), δ 4.10 (t, J=5.1 Hz, 2H), δ 3.28 (m,2H), δ 1.16 (t, J=8.7 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 174.6,134.7, 54.3, 44.2, 38.7, 14.1 ppm; IR ν_(max) (cm⁻¹) 3434, 1640; HRMS(ESI) calcd for C₇H₁₃N₅O (M+) 206.1012. found 206.1014.

1-ethyl-3-(4-methoxyphenethyl)urea (12g)

Light yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.03 (d, J=8.7 Hz, 2H), δ6.78 (t, J=3.9 Hz, 2H), δ 5.85 (q, J=5.1, 6.3 Hz, 2H), δ 3.73 (s, 3H), δ3.31 (q, J=6.6, 7.2 Hz, 2H), δ 3.12 (m, 2H), δ 2.67 (t, J=7.5 Hz, 2H), δ1.08 (t, J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.6, 158.3,131.7, 129.9, 114.1, 55.4, 42.1, 36.1, 35.1, 15.8 ppm; IR ν_(max) (cm⁻¹)3374, 2977, 2837, 1630; HRMS (ESI) calcd for C₁₂H₁₈N₂O₂ (M+) 245.1260.found 245.1263.

1-ethyl-3-phenethylurea (2g)

White solid. mp=75-77° C. ¹H NMR (300 MHz, CDCl₃) δ 7.22 (m, 5H), δ 5.92(d, J=16.2 Hz, 2H), δ 3.86 (q, J=6.6, 6.0 Hz, 2H), δ 3.14 (m, 2H), δ2.78 (t, J=7.8 Hz, 2H), δ 1.10 (t, J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz,CDCl₃) δ 159.6, 139.7, 129.0, 126.5, 41.9, 37.1, 35.1, 15.9 ppm; IRν_(max) (cm⁻¹) 3359, 2972, 2873, 2239, 1633, 1259; HRMS (ESI) calcd forC₁₁H₁₆N₂O (M+) 193.1335. found 193.1334.

1-ethyl-3-(3-phenylpropyl)urea (4g)

White solid. mp=48° C. ¹H NMR (300 MHz, CDCl₃) δ 7.2 (m, 5H), δ 6.15 (s,1H), δ 6.08 (s, 1H), δ 3.26 (m, 4H), δ 2.69 (t, J=9 Hz, 2H), δ 1.84 (m,2H), δ 1.14 (t, J=7.5 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 160.0,142.0, 128.6, 126.1, 40.0, 35.2, 33.5, 32.4, 15.9 ppm; IR ν_(max) (cm⁻¹)3358, 2971, 2867, 1631; HRMS (ESI) calcd for C₁₂H₁₈N₂O (M+) 207.1492.found 207.1489.

1-(4-aminophenethyl)-3-ethylurea (8g)

Light yellow solid. mp=101-103° C. ¹H NMR (300 MHz, CDCl₃) δ 6.94 (d,J=8.4, 2H), δ 6.58 (d, J=6.0 Hz, 2H), δ 5.05 (t, J=3.3 Hz, 2H), δ 3.59(s, 2H), δ 3.28 (q, J=6.9, 6.9 Hz, 2H), δ 3.10 (m, 2H), δ 2.63 (t, J=6.9Hz, 2H), δ 1.04 (t, J=7.2, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.0,145.0, 129.8, 115.5, 42.1, 35.9, 15.7 ppm; IR ν_(max) (cm⁻¹) 3409, 1635,1517, 1263; HRMS (ESI) calcd for C₁₁H₁₇N₃O (M+) 208.1444. found208.1445.

N-ethylindoline-1-carboxamide (6g)

Light yellow solid. mp=111-113° C. ¹H NMR (300 MHz, CDCl₃) δ 7.90 (d,J=8.1 Hz, 1H), δ 7.11-7.04 (m, 2H), δ 6.82 (t, J=7.5 Hz, 1H), δ 5.09 (s,1H), δ 3.78 (t, J=8.7 Hz, 2H), δ 3.27 (m, 2H), δ 3.02 (t, J=9.0 Hz, 2H),δ 1.15 (t, J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ ppm; IR ν_(max)(cm⁻¹) 3403, 2959, 1646; HRMS (ESI) calcd for C₁₁H₁₄N₂O (M+) 190.1179.found 190.1177.

cyclohexyl 2-(pyridin-2-yl)ethylcarbamate (91)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 8.33 (d, J=4.8 Hz, 1H), δ 7.43(t, J=9.9 Hz, 1H), δ 7.02-6.94 (m, 2H), δ 5.72 (s, 1H), δ 4.45 (d, J=3.6Hz, 1H), δ 3.40 (q, J=6.6, 6.3 Hz, 2H), δ 2.83 (t, J=6.6 Hz, 2H), δ1.702-1.027 (m, 10H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.4, 156.5, 149.1,136.8, 123.6, 121.6, 40.4, 37.9, 32.2, 25.5, 23.9 ppm; IR ν_(max) (cm⁻¹)3944, 3692, 3054, 2987, 1709; HRMS (ESI) calcd for C₁₄H₂₀N₂O₂ (M+)249.1598. found 249.1595.

cyclohexyl 2-(1H-indol-3-yl)ethylcarbamate (10l)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 8.74 (s, 1H), δ 7.65 (d, J=6.9Hz, 1H), δ 7.38 (d, J=7.2 Hz, 1H), δ 7.20 (dd, J=7.2, 7.2 Hz, 2H), δ6.96 (s, 1H), δ 4.94 (s, 1H), δ 4.72 (s, 1H), δ 3.63 (d, J=34.2 Hz, 2H),δ 2.98 (s, 2H), δ 1.91-1.31 (m, 10H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ156.8, 136.8, 127.6, 122.6, 119.5, 112.8, 73.3, 70.6, 41.6, 35.8, 32.4,26.1, 24.5 ppm; IR ν_(max) (cm⁻¹) 3334, 2909, 2991, 1701; HRMS (ESI)calcd for C₁₇H₂₂N₂O₂ (M+) 287.1755. found 287.1752.

(1S,2R,5S)-2-isopropyl-5-methylcyclohexyl 2-(pyridin-2-yl)ethylcarbamate(9k)

Yellow solid. mp=57-59° C. ¹H NMR (300 MHz, CDCl₃) δ 8.37 (d, J=4.5 Hz,1H), δ 7.45 (t, J=5.7 Hz, 1H), δ 6.98 (m, 2H), δ 5.55 (s, 1H), δ 4.40(t, J=3.9 Hz, 1H), δ 3.43 (q, J=6.3 Hz, 2H), δ 2.85 (t, J=6.3 Hz, 2H), δ2.01-0.62 (m, 19H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.5, 156.7, 136.3,123.6, 121.6, 74.2, 47.5, 41.6, 40.4, 37.9, 34.4, 31.5, 26.7, 23.6,22.2, 20.9, 16.6 ppm; IR ν_(max) (cm⁻¹) 3406, 2954, 2868, 1695, 1694,1514, 1260; HRMS (ESI) calcd for C₁₈H₂₈N₂O₂ (M+) 305.2224. found305.2221.

2-isopropyl-5-methylphenyl 2-(1H-indol-3-yl)ethylcarbamate (10i)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 8.71 (s, 1H), δ 7.67 (d, J=7.5,1H), δ 7.37 (d, J=8.1 Hz, 1H), δ 7.25 (t, J=7.2 Hz, 1H), δ 7.20 (t,J=7.2 Hz, 1H), δ 6.96 (s, 1H), δ 4.92 (s, 1H), δ 3.52 (t, J=6.3 Hz, 2H),δ 3.01 (t, J=6.3 Hz, 2H), δ 1.26-1.03 (m, 6H), δ 0.99 (d, J=6.6 Hz, 3H),δ 0.95 (d, J=6.9 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 157.1, 136.8,127.7, 122.6, 119.0, 112.9, 111.7, 74.9, 47.6, 41.8, 34.6, 31.7, 26.5,23.8, 22.4, 21.2, 16.8 ppm; IR ν_(max) (cm⁻¹) 3408, 2962, 2926, 1717,1620, 1502, 1457; HRMS (ESI) calcd for C₂₁H₂₄N₂O₂ (M+) 337.1911. found337.1914.

benzyl 2-(pyridin-2-yl)ethylcarbamate (9c)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 8.34 (d, J=6.6 Hz, 1H), δ 7.46(t, J=7.8 Hz, 1H), δ 6.98 (dd, J=7.8, 7.5 Hz, 2H), δ 5.72 (s, 1H), δ4.45 (d, J=7.2 Hz, 1H), δ 3.42 (q, J=6.6, 6.3 Hz, 2H), δ 2.83 (t, J=6.6Hz, 2H), δ 1.70-1.03 (m, 10H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.4,156.5, 149.1, 136.8, 123.6, 121.6, 72.8, 40.4, 37.86, 32.2, 25.5, 23.9ppm; IR ν_(max) (cm⁻¹) 3435, 2092, 1644, 1261; HRMS (ESI) calcd forC₁₅H₁₆N₂O₂ (M+) 257.1285. found 257.1282.

5-isopropyl-2-methylphenyl 2-(1H-indol-3-yl)ethylcarbamate (10j)

¹H NMR (300 MHz, CDCl₃) δ 8.47 (s, 1H), δ 7.74 (d, J=7.8 Hz, 1H), δ7.37-7.05 (m, 6H), δ 6.95 (s, 1H), δ 5.35 (s, 1H), δ 3.69 (t, J=6.6 Hz,2H), δ 3.10 (t, J=6.6 Hz, 2H), δ 2.96 (m, 1H), δ 2.24 (s, 3H), δ 1.33(d, J=6.6 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 155.24, 149.7, 148.4,136.8, 131.2, 128.2, 124.7, 122.7, 120.6, 119.6, 118.9, 112.5, 42.0,33.9, 26.0, 24.3, 16.1 ppm; IR ν_(max) (cm⁻¹) 3336, 2969, 2926, 1719,1503, 1457; HRMS (ESI) calcd for C₂₁H₂₄N₂O₂ (M+) 337.1911. found337.1914.

5-isopropyl-2-methylphenyl 2-(pyridin-2-yl)ethylcarbamate (9j)

¹H NMR (300 MHz, CDCl₃) δ 8.55 (d, J=3.9 Hz, 1H), δ 7.67 (t, J=7.8 Hz,1H), δ 7.27-6.88 (m, 5H), δ 6.46 (t, J=5.4 Hz, 1H), δ 3.68 (q, J=6.6,6.3 Hz, 2H), δ 3.12 (t, J=6.6 Hz, 2H), δ 2.83 (m, 1H), δ 2.13 (s, 3H), δ1.23 (d, J=5.1 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 158.7, 155.0,149.6, 148.1, 138.4, 131.0, 128.0, 124.5, 123.9, 122.4, 120.4, 40.8,37.0, 33.8, 24.2, 15.9 ppm; IR ν_(max) (cm⁻¹) 3054, 2987, 2306, 1734,1501; HRMS (ESI) calcd for C₁₈H₂₂N₂O₂ (M+) 299.1755. found 299.1753.

2-isopropyl-5-methylphenyl 2-(pyridin-2-yl)ethylcarbamate (9i)

¹H NMR (300 MHz, CDCl₃) δ 8.54 (d, J=4.8 Hz, 1H), δ 7.65 (t, J=7.5 Hz,1H), δ 7.25-6.94 (m, 4H), δ 6.83 (s, 1H), δ 6.47 (s, 1H), δ 3.68 (q,J=6.0, 6.0 Hz, 2H), δ 3.07 (m, 2H), δ 2.27 (s, 3H), δ 1.16 (d, J=6.0 Hz,6H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 158.8, 155.4, 148.4, 138.2, 137.8,136.5, 126.9, 126.5, 124.4, 123.4, 122.3, 40.8, 37.1, 27.1, 23.3, 21.1ppm; IR ν_(max) (cm⁻¹) 3054, 2966, 1735, 1250; HRMS (ESI) calcd forC₁₈H₂₂N₂O₂ (M+) 299.1755. found 299.1759.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl 4-aminophenethylcarbamate (8a)

¹H NMR (300 MHz, CDCl₃) δ 6.93 (d, J=8.1 Hz, 2H), δ 6.60 (d, J=8.4 Hz,2H), δ 4.79 (t, J=5.7 Hz, 1H), δ 4.53 (t, J=3.9 Hz, 1H), δ 3.59 (s, 2H),δ 3.33 (q, J=4.5, 5.7 Hz, 2H), δ 2.65 (t, J=6.6 Hz, 2H), δ 2.03 (d,J=6.9 Hz, 1H), δ 1.87 (t, 6.6 Hz, 1H), δ 1.65 (d, 10.5 Hz, 2H), δ 1.43(s, 3H), δ 1.25 (t, 6.4 Hz, 1H), δ 1.01 (q, J=2.7, 2.7 Hz, 1H), δ 0.92(m. 6H), δ 0.79 (d, J=7.2 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 156.7,145.1, 129.8, 128.9, 115.6, 47.6, 42.6, 41.7, 35.4, 34.5, 31.6, 26.5,23.8, 22.3, 21.1, 16.7 ppm; IR ν_(max) (cm⁻¹) 3442, 2955, 2869, 1698,1626, 1264; HRMS (ESI) calcd for C₁₉H₃₀N₂O₂ (M+) 319.2381. found319.2383.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl 4-methoxyphenethylcarbamate(12a)

White solid. mp=95-98° C. ¹H NMR (300 MHz, CDCl₃) δ 7.09 (d, J=8.7 Hz,2H), δ 6.84 (d, J=8.7 Hz, 2H), δ 4.78 (t, J=5.7 Hz, 1H), δ 4.56 (t, 3.9Hz, 1H), δ 3.74 (s, 3H), δ 3.36 (q, J=6.6, 5.7 Hz, 2H), δ 2.73 (t, 7.2Hz, 2H), δ 1.99 (d, J=4.8 Hz, 1H), δ 1.66 (t, J=10.2 Hz, 1H), δ 1.48 (d,J=3.0 Hz, 2H), δ 1.47 (t, J=3.3 Hz, 1H), δ 1.07 (t, 3.5 Hz, 1H), δ 1.03(q, 3.5, 3.0 Hz, 1H), δ 0.93 (m, 6H), δ 0.80 (d, 7.2 Hz, 3H) ppm; ¹³CNMR (75 MHz, CDCl₃) δ 158.4, 156.7, 131.1, 129.9, 114.2, 55.4, 47.6,41.7, 35.5, 34.6, 31.6, 26.5, 23.8, 22.3, 21.1, 16.7 ppm; IR ν_(max)(cm⁻¹) 3372, 2954, 2869, 1684, 1512, 1455, 1242, 1178, 1127, 1056, 623;HRMS (ESI) calcd for C₂₀H₃₁NO₃ (M+) 356.2196. found 356.2199.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl indoline-1-carboxylate (6a)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.90 (s, 1H), δ 7.26 (m, 2H), δ6.91 (m, 1H), δ 4.75 (s, 1H), δ 3.99 (s, 2H), δ 3.89 (t, 6.6 Hz, 2H), δ2.19 (d, J=8.7 Hz, 1H), δ 1.95 (s, 1H), δ 1.55 (d, J=2.4 Hz, 2H), δ 1.41(s, 2H), δ 1.12 (m, 2H), δ 0.84 (m, 10H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ127.7, 124.9, 122.5, 114.9, 47.8, 41.9, 34.7, 34.6, 31.7, 27.6, 26.7,26.6, 23.8, 22.3, 21.1, 16.8, 16.7 ppm; IR ν_(max) (cm⁻¹) 3428, 2955,2869, 1704, 1604, 1488; HRMS (ESI) calcd for C₁₉H₂₇NO₂ (M+) 324.1934.found 324.1929.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl 4-bromophenethylcarbamate(11a)

White solid. mp=104-106° C. ¹H NMR (300 MHz, CDCl₃) δ 7.38 (d, J=5.7 Hz,2H), δ 7.03 (d, 6.0 Hz, 2H), δ 4.74 (s, 1H), δ 4.51 (t, J=2.7 Hz, 1H),63.35 (d, J=4.5 Hz, 2H), δ 2.73 (s, 2H), δ 1.99 (d, J=9.0 Hz, 1H), δ1.64 (t, J=1.5 Hz, 1H), δ 1.63 (t, J=2.4 Hz, 2H), δ 1.41 (m, 1H), δ 1.04(t, J=3.8 Hz, 1H), δ 1.03 (q, J=2.4 Hz, 2H), δ 0.97 (m, 6H), δ 0.76 (d,J=5.1 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 1.56.6, 138.1, 131.8,130.8, 120.5, 74.7, 47.6, 42.1, 41.7, 35.8, 34.5, 31.6, 26.5, 23.8,22.3, 21.0, 16.7 ppm; IR ν_(max) (cm⁻¹) 3364, 2953, 2856, 1684, 1256;HRMS (ESI) calcd for C₁₉H₂₈BrNO₂ (M+) 404.1196. found 404.1191.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl3,4-dihydroquinoline-1(2H)-carboxylate (7a)

Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.76 (d, J=8.4 Hz, 1H), δ 7.18(t, J=1.5 Hz, 1H), δ 7.13 (d, J=1.8 Hz, 1H), δ 6.97, J=0.9 Hz, 1H), δ4.77 (t, J=4.2 Hz, 1H), δ 3.77 (t, J=0.9 Hz, 2H), δ 2.78 (t, J=6.6 Hz,2H), δ 1.18 (d, J=3.0 Hz, 1H), δ 1.99 (m, 3H), δ 1.68 (d, J=2.7 Hz, 2H),δ 1.414 (m, 2H), δ 1.13 (m, 2H), δ 1.10 (m, 6H), δ 0.87 (d, J=7.2 Hz,3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 154.9, 138.7, 129.9, 128.8, 126.1,124.2, 123.6, 76.5, 47.5, 44.9, 41.7, 34.6, 31.7, 27.7, 26.7, 23.8,23.8, 22.3, 21.1, 16.7 ppm; IR ν_(max) (cm⁻¹) 3434, 2954, 2870, 2105,1694; HRMS (ESI) calcd for C₂₀H₂₉NO₂ (M+) 338.2091. found 338.2088.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl phenethylcarbamate (2a)

White solid. mp=84-86° C. ¹H NMR (300 MHz, CDCl₃) δ 7.29 (m, 5H), δ 4.91(t, J=4.2 Hz, 1H), δ 4.58 (s, 1H), δ 3.39 (s, 2H), δ 2.79 (t, J=7.8 Hz,2H), δ 2.05 (d, J=8.7 Hz, 1H), δ 1.93 (t, J=4.2 Hz, 1H), δ 1.66 (s, 2H),δ 1.47 (s, 1H), δ 1.28 (t, J=3.8 Hz, 1H), δ 1.04 (q, 10.2, 9.3 Hz, 2H),δ 0.91 (m, 8H), δ 0.80 (d, J=0.60, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ156.7, 139.2, 129.1, 128.9, 128.8, 128.7, 126.6, 126.5, 74.6, 47.6,45.3, 42.4, 41.9, 41.8, 36.5, 34.9, 31.6, 26.5, 23.8, 23.4, 22.6, 22.3,21.1, 16.4 ppm; IR ν_(max) (cm⁻¹) 3360, 2957, 1682, 1526, 1259, 1024,798; HRMS (ESI) calcd for C₁₉H₂₉NO₂ (M+) 343.2381. found 343.2383.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl2,3-dihydro-1H-inden-2-ylcarbamate (3a)

White solid. mp=167-170° C. ¹H NMR (300 MHz, CDCl₃) δ 7.19 (m, 4H), δ4.99 (s, 1H), δ 4.57 (m, 2H), δ 3.25 (m, 3H), δ 2.75 (m, 2H), δ 2.02 (d,J=11.7 Hz, 2H), δ 1.94 (t, J=2.1 Hz, 1H), δ 1.67 (d, J=12.9, 2H), δ 1.58(s, 1H), δ 1.45 (t, J=3.3 Hz, 1H), 1.05 (q, J=3.0, 7.5 Hz, 2H), δ 0.93(m, 6H), δ 0.78 (d, J=7.2 Hz, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.6,126.9, 126.8, 124.9, 124.8, 51.6, 50.3, 47.6, 45.3, 41.7, 40.8, 40.6,34.8, 34.5, 31.0, 31.6, 26.5, 23.7, 23.4, 22.5, 22.3, 21.3, 21.1, 16.6,16.3 ppm; IR ν_(max) (cm⁻¹) 3409, 1688; HRMS (ESI) calcd for C₂₀H₂₉NO₂(M+) 338.2091. found 338.2098.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl 2-(pyridin-2-yl)ethylcarbamate(9a)

White solid. mp=59-63° C. ¹H NMR (300 MHz, CDCl₃) δ 8.50 (q, J=0.6, 2.4Hz, 1H), δ 7.61 (t, 2.1 Hz, 1H), δ 7.13 (m, 2H), δ 5.75 (s, 1H), δ 4.54(t, J=7.2 Hz, 1H), δ 3.59 (q, J=5.7, 6.3 Hz, 2H), δ 2.99 (t, J=6.3 Hz,2H), δ 2.03 (d, J=11.7 Hz, 1H), δ 1.89 (m, 1H), δ 1.59 (tm, 2H), δ 1.27(s, 1H), δ 1.27 (t, J=10.8 Hz, 1H), δ 0.97 (m, 2H), δ 0.91 (m, 6H), δ0.76 (d, J=6.9 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.5, 156.6,140.3, 136.5, 123.6, 121.5, 74.2, 71.1, 50.1, 47.5, 41.6, 40.4, 37.9,34.7, 31.8, 31.4, 26.3, 23.6, 20.9, 16.2 ppm; IR ν_(max) (cm⁻¹) 3406,2954, 2868, 1695, 1694, 1514, 1260; HRMS (ESI) calcd for C₁₈H₂₈N₂O₂ (M+)305.2224. found 305.2229.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl2-(2H-1,2,3-triazol-2-yl)ethylcarbamate (5a)

White residue. ¹H NMR (300 MHz, CDCl₃) δ 7.59 (s, 2H), δ 5.11 (s, 1H), δ4.54 (s, 2H), δ 3.72 (s, 2H), δ 3.24 (s, 1H), δ 1.90 (m, 2H), δ 1.61 (s,1H), δ 1.52 (s, 2H), δ 1.45 (s, 1H), δ 1.24 (s, 1H), 1.09 (m, 2H), δ1.05 (m, 8H), δ 0.76 (d, J=4.2 Hz, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ156.5, 134.6, 74.8, 71.7, 54.7, 50.3, 47.7, 47.5, 41.8, 40.5, 34.5,31.6, 26.5, 23.7, 22.2, 16.3 ppm; IR ν_(max) (cm⁻¹) 3434, 2955, 2088,1642, 1256; HRMS (ESI) calcd for C₁₅H₂₆N₄O₂ (M+) 304.4125. found304.4129.

(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl 3-phenylpropylcarbamate (4a)

White solid. mp=75-78° C. ¹H NMR (300 MHz, CDCl₃) δ 7.28 (t, J=7.8 Hz,2H), δ 7.16 (d, J=7.2 Hz, 2H), δ 4.86 (s, 1H), δ 4.55 (t, J=6.9 Hz, 1H),δ 3.201 (q, J=6.6, 6.0 Hz, 2H), δ 2.64 (t, J=7.5 Hz, 2H), δ 2.05 (d,J=11.7 Hz, 1H), δ 1.95 (t, J=2.7 Hz, 1H), δ 1.89 (m, 2H), δ 1.66 (d,J=10.8 Hz, 2H), δ 1.48 (s, 1H), δ 1.06 (t, J=8.4 Hz, 1H), δ 1.06 (m,2H), δ 1.02 (m, 8H), δ 0.80 (d, J=6.9 Hz, 3H) ppm; ¹³C NMR (75 MHz,CDCl₃) δ 156.9, 141.8, 128.7, 128.6, 126.2, 126.1, 74.6, 47.4, 41.8,40.1, 34.6, 33.5, 33.3, 31.9, 31.6, 26.6, 23.8, 22.4, 16.8 ppm; IRν_(max) (cm⁻¹) 3410, 3057, 2960, 1713, 1421, 1362, 1267, 1222, 191, 846,736, 702; HRMS (ESI) calcd for C₂₀H₃₁NO₂ (M+) 340.2247. found 340.2241.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl2-(2H-1,2,3-triazol-2-yl)ethylcarbamate (5f)

White solid. mp=135-139° C. ¹H NMR (300 MHz, CDCl₃) δ 7.58 (s, 2H), δ5.31 (t, 6.9 Hz, 1H), δ 4.53 (t, J=5.1 Hz, 1H), δ 3.69 (q, J=5.1 Hz, 2H)δ 3.47 (m, 2H), δ 2.49 (s, 1H), δ 2.45 (q, J=9.3, 4.2 Hz, 2H), δ 1.96(m, 6H), δ 1.54-1.30 (m, 11H), δ 1.25-1.09 (m, 14H), 61.01-0.82 (m,10H), 60.65 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ141.1, 134.6, 121.8,71.8, 57.0, 56.9, 50.3, 42.5, 40.0, 39.8, 37.5, 36.7, 36.4, 36.1, 32.1,31.8, 28.5, 28.2, 24.5, 24.1, 23.1, 229, 21.3, 19.6, 19.5, 18.9, 12.1ppm; IR ν_(max) (cm⁻¹) 3398, 2037, 1637; HRMS (ESI) calcd for C₃₂H₅₂N₄O₂(M+) 547.3982. found 547.3981.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl4-aminophenethylcarbamate (8f)

Yellow hygroscopic solid. ¹H NMR (300 MHz, CDCl₃) δ 6.93 (d, J=7.5 Hz,2H), δ 6.588 (d, J=7.5 Hz, 2H), δ 5.35 (m, 1H), δ 4.92 (s, 1H), δ 4.47(m, 1H), 63.32 (d, J=6.0 Hz, 2H), δ 2.54 (t, J=6.6 Hz, 2H), δ 2.31 (m,2H), δ 2.02-1.98 (m, 5H), δ 1.54-1.27 (m, 11H), δ 1.10-0.85 (m, 24H), δ0.67 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 156.4, 145.2, 141.2, 140.1,129.8, 128.8, 122.7, 115.6, 74.4, 57.-, 56.9, 56.4, 50.2, 42.6, 40.0,39.8, 38.9, 37.3, 36.8, 36.5, 35.5, 32.1, 31.8, 28.4, 28.3, 24.5, 24.2,23.2, 22.0, 21.3, 10.7, 19.5, 19.0, 12.1 ppm; IR ν_(max) (cm⁻¹) 3407,2937, 2868, 2245, 1695, 1631, 1516; HRMS (ESI) calcd for C₃₆H₅₆N₂O₂ (M+)549.4415. found 549.4420.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl2,3-dihydro-1H-inden-2-ylcarbamate (3f)

mp=114-116° C. ¹H NMR (300 MHz, CDCl₃) δ 7.19 (m, 4H), δ 5.37 (d, J=6.6Hz, 1H), δ 5.15 (m, 1H), δ 4.52 (s, 1H), δ 3.40 (m, 1H), δ 3.24 (m, 2H),δ 2.78 (m, 2H), δ 2.27 (m, 3H), δ 2.04-1.84 (m, 5H), δ 1.55-1.26 (m,11H), δ 1.12-0.87 (m, 23H), δ 0.69 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃)δ 141.4, 126.9, 125.0, 124.9, 122.8, 121.89, 71.9, 57.0, 56.9, 56.4,52.4, 51.7, 50.4, 50.2, 42.5, 42.5, 40.8, 40.5, 40.1, 40.0, 39.8, 37.6,36.8, 36.5, 36.1, 32.2, 32.1, 31.8, 28.5, 28.5, 24.6, 24.2, 23.1, 22.9,21.4, 21.3, 19.7, 19.6, 19.0, 12.1 ppm; IR ν_(max) (cm⁻¹) 3358, 2937,2867, 1693, 1551, 1466; HRMS (ESI) calcd for C₃₇H₅₅NO₂ (M+) 568.4125.found 568.4136.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl4-bromophenethylcarbamate (11f)

White solid. mp=142-145° C. ¹H NMR (300 MHz, CDCl₃) δ 7.37 (d, J=8.4 Hz,2H), δ 6.99 (d, J=8.4 Hz, 2H), δ 5.34 (s, 1H), δ 4.98 (t, J=5.7, 1H), δ4.44 (m, 1H), δ 3.34 (q, J=6.6 Hz, 1H), δ 2.72 (t, J=6.6 Hz, 2H), δ 2.27(m, 2H), δ 2.32-1.79 (m, 5H), δ 1.52-1.34 (m, 11H), δ 1.22-0.86 (m,22H), δ 0.65 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 202.3, 156.3, 139.1,138.1, 131.9, 131.7, 130.8, 122.8, 120.5, 74.5, 56.9, 57.4, 50.2, 42.5,42.1, 39.9, 39.8, 38.8, 37.3, 36.8, 36.5, 36.1, 32.1, 29.6, 28.4, 28.2,24.5, 24.2, 23.1, 22.9, 21.3, 19.6, 19.0, 12.1 ppm; IR ν_(max) (cm⁻¹)3435, 2947, 2867, 2249, 1793, 1488; HRMS (ESI) calcd for C₃₆H₅₄BrNO₂(M+) 634.3230. found 634.3210.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylindoline-1-carboxylate (6f)

White solid, mp=157-159° C. ¹H NMR (300 MHz, CDCl₃) δ 7.86 (s, 1H), δ7.16 (t, 2H), δ 6.93 (t, J=7.5 Hz, 1H), δ 5.44 (d, J=4.5 Hz, 1H), δ 4.68(s, 1H), δ 3.98 (t, J=7.5 Hz, 2H), δ 3.07 (t, J=8.7 Hz, 2H), δ 2.07-1.84(m, 5H), δ 1.59-1.28 (m, 11H), δ 1.21-1.04 (m, 10H), δ 1.03-0.90 (m,12H), δ 0.72 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 202.3, 149.9, 139.9,127.7, 122.9, 122.6, 115.1, 75.1, 56.9, 56.5, 50.3, 47.6, 42.6, 40.0,39.8, 37.7, 37.3, 36.8, 36.8, 36.4, 32.2, 32.1, 31.8, 28.6, 28.3, 27.6,24.6, 24.3, 23.2, 22.9, 21.4, 19.7, 19.0, 12.2 ppm; IR ν_(max) (cm⁻¹)3444, 2944, 1699, 1604; HRMS (ESI) calcd for C₃₆H₅₃NO₂ (M+) 532.4150.found 532.4152.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl4-methoxyphenethylcarbamate (12f)

White residue. ¹H NMR (300 MHz, CDCl₃) δ 7.10 (d, J=6.6 Hz, 2H), δ 6.83(d, J=6.6 Hz, 2H), δ 5.37 (d, J=4.8 Hz, 1H), δ 4.88 (s, 1H), δ 4.48 (m,1H), δ 3.75 (s, 3H), δ 3.36 (q, J=6.0, 6.6 Hz, 2H), δ 2.72 (t, J=6.9 Hz,2H), δ 2.32 (m, 2H), δ 1.98-1.81 (m, 5H), δ 1.52-1.36 (m, 11H), δ1.28-1.13 (m, 12H), δ 1.11-0.86 (m, 10H), δ 0.67 (s, 3H) ppm; ¹³C NMR(75 MHz, CDCl₃) δ 158.5, 156.4, 140.0, 131.1, 129.9, 122.7, 114.2,114.1, 74.5, 56.9, 56.4, 55.4, 50.2, 42.5, 39.9, 39.8, 38.8, 37.3, 36.8,36.5, 36.1, 35.5, 32.1, 28.5, 28.4, 28.3, 24.5, 24.1, 23.1, 22.9, 21.3,19.6, 18.9, 12.1 ppm; IR ν_(max) (cm⁻¹) 3418, 2937, 2867, 1695, 1512,1465, 1246, 733; HRMS (ESI) calcd for C₃₇H₅₇NO₃ (M+) 586.4231. found586.4230.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylphenethylcarbamate (2f)

White solid. mp=74-76° C. ¹H NMR (300 MHz, CDCl₃) δ 7.39 (t, J=1.2 Hz,2H), δ 7.18 (m, 3H), δ 5.36 (d, J=2.7 Hz, 1H), δ 4.83 (s, 1H), δ 4.48(m, 1H), δ 3.39 (d, J=4.5 Hz, 2H), δ 2.33 (m, 2H), δ 2.02-1.82 (m, 5H),δ 1.55-1.33 (m, 18H), δ 1.12-1.03 (m, 7H), δ 0.96-0.86 (m, 9H), δ 0.67(s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 156.36, 140.1, 129.1, 129.0,128.8, 128.7, 126.7, 122.7, 56.0, 56.5, 50.3, 42.6, 42.4, 40.0, 39.8,38.9, 37.3, 36.8, 36.5, 36.1, 32.2, 32.1, 28.5, 28.5, 28.3, 24.6, 23.2,22.9, 21.3, 10.6, 10.0, 12.1 ppm; IR ν_(max) (cm⁻¹) 3419, 2944, 2867,2089, 1694, 1255, 1137; HRMS (ESI) calcd for C₃₆H₅₅NO₂ (M+) 556.4125.found 556.4128.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-phenylpropylcarbamate (4f)

White solid. mp=87-89° C. ¹H NMR (300 MHz, CDCl₃) δ 7.24 (t, J=3.3 Hz,2H), δ 7.25 (t, J=5.4 Hz, 3H), δ 5.35 (d, J=3.6 Hz, 1H), δ 4.89 (t,J=4.5 Hz, 1H), δ 4.49 (m, 1H), δ 3.17 (d, J=4.8 Hz, 2H), δ 2.35 (m, 2H),δ 2.01-1.78 (m, 8H), δ 1.55=1.41 (m, 11H), δ 1.29-1.12 (m, 12H), δ1.05-0.91 (m, 11H), δ 0.67 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 156.5,141.7, 140.1, 128.7, 128.6, 126.2, 122.7, 74.4, 56.9, 56.5, 50.3, 42.6,50.7, 50.0, 39.8, 38.9, 37.3, 36.9, 36.5, 35.1, 33.4, 32.1, 31.9, 28.5,28.4, 28.3, 24.5, 24.2, 23.2, 22.9, 21.3, 19.6, 19.0, 12.2 ppm; IRν_(max) (cm⁻¹) 3419, 2944, 2867, 2089, 1694, 1255, 1137; HRMS (ESI)calcd for C₃₇H₅₇NO₂ (M+) 570.4282. found 570.4287.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl2-(pyridin-2-yl)ethylcarbamate (9f)

White residue. ¹H NMR (300 MHz, CDCl₃) δ 8.50 (d, J=3.6 Hz, 1H), δ 7.59(t, 0.9 Hz, 1H), δ 5.7 (d, J=5.7 Hz, 1H), δ 7.09 (t, J=5.7 Hz, 1H), δ5.73 (s, 1H), δ 5.35 (s, 1H), δ 4.49 (s, 1H), δ 3.58 (d, J=4.2 Hz, 2H),δ 2.99 (t, J=4.8 Hz, 2H), δ 2.34 (m, 2H), δ 2.02-1.81 (m, 5H), δ1.54-1.34 (m, 10H), δ 1.19-1.08 (m, 11H), δ 1.06-0.86 (m, 14H), δ 0.68(s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 159.6, 156.3, 149.4, 140.0,136.6, 123.6, 122.5, 121.6, 74.2, 56.9, 56.4, 50.2, 42.5, 40.4, 39.9,39.7, 38.8, 37.0, 36.7, 36.4, 32.1, 32.0, 28.4, 28.4, 28.2, 24.5, 24.1,23.0, 22.8, 21.2, 19.5, 18.9, 12.1 ppm; IR ν_(max) (cm⁻¹) 3434, 2938,2869, 2094, 1708, 1641, 1264; HRMS (ESI) calcd for C₃₅H₅₄N₂O₂ (M+)535.4259. found 535.4257.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3,4-dihydroquinoline-1(2H)-carboxylate (7f)

White solid. mp=107-110° C. ¹H NMR (300 MHz, CDCl₃) δ 7.71 (d, J=8.1 Hz,1H), δ 7.18 (t, J=7.5 Hz, 1H), δ 7.09 (d, J=7.2 Hz, 1H), δ 6.99 (t,J=7.5 Hz, 1H), δ 5.41 (d, J=4.21 Hz, 1H), δ 4.65 (m, 1H), δ 3.76 (t,J=5.7 Hz, 2H), δ 2.77 (t, J=6.6 Hz, 2H), δ 2.45 (m, 2H), δ 2.04-1.89 (m,7H), δ 1.86-1.55 (m, 8H), δ 1.44-1.15 (m, 10H), δ 1.12-1.04 (m, 9H), δ0.94-0.88 (m, 9H), δ 0.69 (s, 3H), ¹³C NMR (75 MHz, CDCl₃) δ 154.6,140.0, 138.6, 130.1, 128.8, 126.1, 124.2, 123.6, 122.8, 75.9, 56.9,56.4, 40.3, 44.9, 42.6, 39.9, 39.8, 38.8, 37.3, 36.9, 36.4, 36.1, 32.2,32.1, 28.5, 28.4, 28.3, 27.7, 24.5, 24.1, 23.7, 23.1, 22.1, 21.3, 19.5,18.9, 12.1 ppm; IR ν_(max) (cm⁻¹) 3408, 2962, 2927, 1717, 1620, 1602,1457; HRMS (ESI) calcd for C₃₇H₅₅NO₂ (M+) 568.4125. found 568.4127.

(3S,8S,9S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl2-(1H-indol-3-yl)ethylcarbamate (10f)

Light yellow solid. mp=149-152° C. ¹H NMR (300 MHz, CDCl₃) δ 8.57 (s,1H), δ 7.54 (d, J=7.2 Hz, 1H), δ 7.26 (d, J=7.2 Hz, 1H), δ 7.10 (m, 2H),δ 6.79 (s, 1H), δ 5.31 (s, 1H), δ 4.90 (s, 1H), δ 4.52 (s, 1H), δ 3.41(s, 2H), δ 2.87 (s, 2H, δ 2.27 (s, 2H), δ 1.99-1.77 (m, 5H), δ 1.52-1.34(m, 11H), δ 1.12-0.88 (m, 22H), δ 0.66 (s, 3H) ppm; ¹³C NMR (75 MHz,CDCl₃) δ 156.8, 140.0, 136.8, 127.6, 122.9, 122.6, 122.2, 119.5, 118.9,112.8, 111.8, 74.7, 56.9, 56.5, 50.3, 42.6, 41.7, 40.1, 39.9, 38.9,37.3, 36.8, 36.5, 36.2, 32.2, 28.6, 28.4, 24.7, 23.3, 22.9, 21.4, 19.7,19.2, 12.2 ppm; IR ν_(max) (cm⁻¹) 3412, 3056, 2945, 2868, 2247, 1697,1515; HRMS (ESI) calcd for C₃₆H₅₄N₂O₂ (M+) 547.4259. found 547.4257.

BIOLOGY EXPERIMENTAL

Procedure to Determine the Inhibitory Effect of Test Compounds.

Inhibition assays were performed by taking an overnight culture ofbacterial strain and subculturing it at an OD₆₀₀ of 0.01 into themedium. Stock solutions of predetermined concentrations of the testcompound were then made in the medium. These stock solutions werealiquoted (100 μL) into the wells of the 96-well PVC microtiter plate.Sample plates were then wrapped in GLAD Press n'Seal® followed by anincubation under stationary conditions for 24 h at 37° C. Afterincubation, the medium was discarded from the wells and the plates werewashed thoroughly with water. Plates were then stained with 100 μL of0.1% solution of crystal violet (CV) and then incubated at ambienttemperature for 30 min. Plates were washed with water again and theremaining stain was solubilized with 200 μL of 95% ethanol. A sample of125 μL of solubilized CV stain from each well was transferred to thecorresponding wells of a polystyrene microtiter dish. Biofilm inhibitionwas quantitated by measuring the OD₅₄₀ of each well in which a negativecontrol lane wherein no biofilm was formed served as a background andwas subtracted out.

Colony Count Procedure to Determine the Effect of Leading Test Compoundson MRSA and E. Coli Planktonic Viability:

Colony counts were performed by taking an overnight culture of bacterialstrain and subculturing it at an OD₆₀₀ of 0.01 into the necessary medium(tryptic soy broth with a 0.5% glucose supplement (TSBG) for MRSA,Luria-Bertani (LB) medium for E. coli). The resulting bacterialsuspension was then aliquoted (3.0 mL) into culture tubes. A testcompound was then added to the medium of the test samples at apredetermined concentration to the medium of the test samples. Controlswere employed in which no test compound was added to the bacterialsuspension. Samples were then placed in an incubator at 37° C. andshaken at 200 rpm until the OD₆₀₀ of the control samples reachedapproximately 1.2. At this point, 100 μL was taken from each culturetube and then diluted serially into LB medium. Then, 10 μL was removedfrom each serial dilution and plated out on a square gridded Petri dishfollowed by 16 h of incubation at 37° C. to grow viable colonies, whichwere quantified through employment of the track-dilution method.

Red Blood Cell Hemolysis Assay.

Hemolysis assays were performed on mechanically difibrinated sheep blood(Hemostat Labs: DSB100). 1.5 mL of blood was placed into amicrocentrifuge tube and centrifuged at 10000 rpm for ten minutes. Thesupernatant was removed and then the cells were resuspended with 1 mL ofphosphate-buffered saline (PBS). The suspension was centrifuged, thesupernatant was removed and cells resuspended two more times. The finalcell suspension was then diluted tenfold. Test compound solutions weremade in PBS in small culture tubes and then added to aliquots of thetenfold suspension dilution. PBS alone was used as a negative controland as a zero hemolysis marker whereas a 1% Triton X sample was used asa positive control and the 100% lysis marker. Samples were then placedin an incubator at 37° C. while being shaken at 200 rpm for one hour.After one hour, the samples were transferred to microcentrifuge tubesand then centrifuged at 10000 rpm for ten minutes. The resultingsupernatant was diluted by a factor of 40 in distilled water. Theabsorbance of the supernatant was measured with a UV spectrometer at a540 nm wavelength.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A compound of Formula (I)(b):

wherein: R¹ is an amine-substituted aryl, or R¹ is a heteroaryl selectedfrom the group consisting of: indazolyl, indolizinyl, isoindolyl,isoquinolyl, isothiazolyl, naphthyridinyl, phthalazinyl, purinyl,pyrazinyl, pyridazinyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl,pyrimidinyl, pyrrolyl, quinolyl, quinoxalinyl and triazolyl; n=2 to 10,saturated or unsaturated; each occurrence of R^(x) and R^(y) is presentor absent (depending upon chain saturation), and is each independently Hor alkyl; and R² is selected from the group consisting of: H, alkyl,alkenyl and alkynyl, or a pharmaceutically acceptable salt thereof. 2.The compound of claim 1, wherein R¹ is selected from the groupconsisting of:


3. The compound of claim 1, wherein said compound is a compound ofFormula (I)(a)(i):

wherein: n=2 to 10, saturated or unsaturated; each occurrence of R^(x)and R^(y) is present or absent (depending upon chain saturation), and iseach independently H or alkyl; and R² is selected from the groupconsisting of: H and alkyl, or a pharmaceutically acceptable saltthereof.
 4. The compound of claim 1, wherein said compound is a compoundof Formula (I)(a)(ii):

wherein: n=2 to 10, saturated or unsaturated; each occurrence of R^(x)and R^(y) is present or absent (depending upon chain saturation), and iseach independently H or alkyl; and R² is selected from the groupconsisting of: H and alkyl, or a pharmaceutically acceptable saltthereof.
 5. The compound of claim 1, wherein n=2 to
 5. 6. The compoundof claim 1, wherein n=2 to 5, saturated.
 7. The compound of claim 1,wherein said compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 8. A compositioncomprising a carrier and an effective amount of the compound of claim 1.9. A composition comprising the compound of claim 1 and a biocide.